Alternatives to antibiotic growth promoters have been widely exploited due to concerns about antimicrobial resistance. These feed additives improve growth, in part, by modulating intestinal microbiota. However, their impact on male reproductive performance is not well elucidated. Therefore, the objective of this study was to evaluate the impacts of a yeast fermentation product (YP) and Bacillus subtilis on rooster semen quality and microbiota. Dietary supplementation of YP linearly increased the concentration of yeast and bacteria in semen, whereas it linearly decreased sperm motility, suggesting that bacteria attached to yeast were excreted from the gut, contaminated semen at the cloaca and then decreased sperm movement. However, direct in vitro exposure of semen or dietary supplementation with B. subtilis did not affect semen quality or seminal concentration of this bacterium, likely because Bacillus naturally occur in semen. In conclusion, unlike B. subtilis, dietary YP can alter semen quality by altering semen microbiota.