<p dir="ltr">Healthcare-acquired infections (HAIs) cause a burden in acute care hospitals in the United States. HAIs are caused by <i>Staphylococcus aureus</i>, along with other pathogens found on high-touch and non-high-touch surfaces in hospital environments (e.g., bed rails, blood pressure cuffs, countertops, and floors). To minimize the growth and cross-contamination of pathogens, it is vital to use disinfectants for surface decontamination. In this work, the impact of different application methods and disinfectant active agents was evaluated for use on different healthcare-related surfaces. The first study examined the cross-contamination potential of an auto-scrubber when used to clean and disinfect a 2m² vinyl floor contaminated with <i>S. aureus</i>. Five EPA-registered disinfectants and a cleaner were used with three application methods. Hydrogen peroxide and quaternary ammonium compounds-based disinfectants significantly resulted in less cross-contamination compared to the cleaner. However, there were no significant differences among the application methods used. In the second study, manual floor cleaning and disinfecting on a two-square-meter vinyl floor with three different moping materials were evaluated to assess their ability to prevent cross-contamination. Evidence showed that there were significant differences among the products used, with Hydrogen peroxide and quaternary ammonium compound products being the most effective. The cleaner caused the most cross-contamination, while cotton mops resulted in significant cross-contamination among materials used. Study three investigated the differences among four application methods used with three different wiping cloths (Cotton, microfiber, and nonwoven) on a 2m² Formica board. A spray surface and wipe method was successful in decreasing <i>S. aureus</i> on the surface. A hydrogen peroxide-based product was most effective in reducing bacteria at contamination areas and minimizing cross-contamination. Microfiber cloth picked up significantly more bacteria at contaminated areas. Post disinfection, there was evidence of cross-contamination at sampling areas regardless of product type, wiping cloth, and application methods used, with the cotton cloth causing the most cross-contamination. Viable bacteria were found on the wiping cloths used and on worker's gloves. Together, this work shows that the use of disinfectants is important in hospital environments. The choice of product, wiping material, and application method are principal in the disinfection process as they influence disinfection failure or success.</p>