Objectives: Mouthrinses are widely used by dental patients in daily oral hygiene practices. However, claims of antimicrobial effects are often made by mouthrinse manufacturers without substantiation by laboratory testing on complex oral microbial communities, such as the dorsal tongue microbiota, which are readily exposed in vivo to mouthrinse solutions, and contribute to the etiology of oral halitosis (bad breath). This study employed a modified Kirby-Bauer zone of inhibition test to comparatively examine the in vitro antimicrobial activity of 12 commercial mouthrinse formulations on a mixture of three bacterial species frequently isolated from the human tongue dorsum. Methods: The 12 commercial mouthrinse formulations tested were 1.) Perio-Aid Treatment Mouthwash (Dentaid S.L., Cerdanyola del Vallès, Spain; containing 0.12% chlorhexidine gluconate and 0.05% cetylpyridinium chloride without alcohol), 2.) Paroex (Sunstar Americas, Inc., Schaumburg, IL; containing 0.12% chlorhexidine gluconate without alcohol), 3.) Peridex (3M ESPE Dental Products, St. Paul, MN, USA; containing 0.12% chlorhexidine gluconate plus 11.6% alcohol), 4.) Perio-Aid Maintenance Mouthwash (Dentaid S.L.; containing 0.05% chlorhexidine gluconate and 0.05% cetylpyridinium chloride without alcohol), 5.) Halita Mouthwash for Halitosis (Dentaid S.L.; containing 0.05% chlorhexidine gluconate, 0.05% cetylpyridinium chloride, and 0.14% zinc lactate without alcohol), 6.) Crest Pro-Health Clinical (Procter & Gamble, Cincinnati, OH, USA; containing 0.1% cetylpyridinium chloride without alcohol), 7.) Therasol (formerly from OraTec Corp., Manassas, VA; containing C31G complex of alkyl dimethyl amine oxide and alkyl dimethyl glycine plus 8% alcohol), 8.) Listerine Cool Mint Zero Alcohol (Johnson & Johnson Consumer, Inc., Skillman, NJ, USA; containing essential oils 0.092% eucalyptol, 0.042% menthol, 0.06% methyl salicylate, and 0.064% thymol, without alcohol), 9.) PerioShield Oral Health Rinse (formerly from Sunstar Americas, Inc.; containing 0.2% delmopinol hydrochloride plus 1.5% alcohol), 10.) Listerine Cool Mint (Johnson & Johnson Consumer, Inc.; containing essential oils 0.092% eucalyptol, 0.042% menthol, 0.06% methyl salicylate, and 0.064% thymol, plus 21.6% alcohol), 11.) CloSys Antiseptic Oral Rinse (Rowpar Pharmaceuticals, Scottsdale, AZ, containing stabilized chlorine dioxide without alcohol), and 12.) PerioMed Antimicrobial Oral Rinse (3M ESPE Dental Products; containing 0.63% stannous fluoride without alcohol). Streptococcus salivarius subsp. salivarius ATCC 13419, Veillonella atypica ATCC 17744, and Prevotella melaninogenica ATCC 25845, which are among the most frequent bacterial isolates from the human tongue dorsum, were grown on enriched Brucella blood agar, comprised of 4.3% Brucella agar supplemented with 0.3% bacto-agar, 5% defibrinated sheep blood, 0.2% hemolyzed sheep red blood cells, 0.0005% hemin, and 0.00005% menadione. Pure cell suspensions of each species were adjusted to a 0.5 McFarland turbidity standard (approximately 1.5 x 108 CFU/ml), and combined equally into a standardized mixture. Undiluted 0.1 ml aliquots of the standardized bacterial mixture were spread with sterile cotton-tipped swabs onto non- selective enriched Brucella blood agar culture plates. After inoculation, four 7-mm diameter cylindrical wells were punched into each of the culture plates and filled with 60 μl of one of the commercial mouthrinse formulations, or sterile saline as a negative control. The inoculated culture plates were incubated upright in anaerobic jars containing 85% N2-10% H2-5% CO2 at 37 °C for four days, after which the diameter of inhibition zones against the standardized bacterial mixture at each well was measured at three locations to the nearest millimeter with a Boley gauge. Differences in mean bacterial inhibition zones (after subtraction of the agar well diameter) among the mouthrinse formulations and sterile saline were evaluated using a one-way analysis-of-variance and a post-hoc Tukey honestly significant difference test, with a Bonferroni adjustment for multiple comparisons. A P-value of < 0.05 was required for statistical significance. Results: Perio-Aid Treatment exhibited significantly greater in vitro antimicrobial inhibition than the other tested mouthrinse formulations and sterile saline, followed in descending in vitro antibacterial activity by Paroex, Peridex, Perio-Aid Maintenance, Halita, Crest Pro-Health Clinical, Therasol, Listerine Cool Mint Zero Alcohol, and PerioShield. Listerine Cool Mint with alcohol, Closys, and PerioMed were not significantly different from sterile saline in antibacterial in vitro activity. Conclusions: The mouthrinse containing 0.12% chlorhexidine gluconate plus 0.05% cetylpyridinium chloride (Perio-Aid Treatment) exerted the greatest in vitro inhibitory potential against a combination of three bacterial species frequently predominant on the human tongue dorsum. Significantly less antibacterial effects were found with chlorhexidine gluconate or cetylpyridinium chloride alone, or chlorhexidine gluconate at lower concentrations in combination with cetylpyridinium chloride. The relative lack of in vitro antibacterial activity of mouthrinses comprised of essential oils with alcohol, stabilized chlorine dioxide, or stannous fluoride raises questions about their potential clinical effectiveness against dorsal tongue surface biofilms and oral halitosis. / Oral Biology