Background: The risk of SSI increases in the presence of foreign materials and may be caused by organisms with low pathogenicity, such as skin flora derived from hands of surgical team members in the event of a glove breach. Previously, we were able to demonstrate that a novel antimicrobial surgical glove coated chlorhexidine-digluconate as the active ingredient on its inner surface was able to suppress surgeons' hand flora during operative procedures by a magnitude of 1.7 log 10 cfu/mL. Because of the clinical design of that study, we were not able to measure the full magnitude of the possible antibacterial suppression effect of antimicrobial gloves over a full 3 h period. Methods: The experimental procedure followed the method for assessment of the 3-h effects of a surgical hand rub's efficacy to reduce the release of hand flora as described in the European Norm EN 12791. Healthy volunteers tested either an antimicrobial surgical glove or non-antimicrobial surgical latex gloves in a standardized laboratory-based experiment over a wear time of 3 h. Results: Wearing antimicrobial surgical glove after a surgical hand rub with 60% (v/v) n-propanol resulted in the highest 3-h reduction factor of 2.67 log 10. Non-antimicrobial surgical gloves demonstrated significantly lower (p ≤ 0.01) 3-h reduction factors at 1.96 log 10 and 1.68 log 10, respectively. Antibacterial surgical gloves are able to maintain a sustainable bacterial reduction on finger tips in a magnitude of almost 3 log 10 (log 10 2.67 cfu) over 3 h wear time. Conclusion: It was demonstrated that wear of an antibacterial surgical glove coated with chlorhexidine-digluconate is able to suppress resident hand flora significantly over a period of 3-h.