INTRODUCTION: Infectious endophthalmitis, a vision-threatening disease caused by exogenous or endogenous microbial invasion, may require vitrectomy with or without silicone oil (SO) tamponade in severe cases. SO antimicrobial effects have been suggested but not demonstrated in an in vitro environment mimicking real clinical conditions. Using an in vitro intraocular tamponade model, we investigated the antimicrobial activity of SO against 11 bacterial and 1 fungal species, including antibiotic-resistant strains of Staphylococcus aureus and Klebsiella pneumoniae. METHODS: Following inoculation, assay plates were coated with SO or balanced salt solution or left uncoated. Colony-forming units were measured 24, 48, and 72 h after incubation and compared among the three media types and three timepoints for each strain. Differences in colony size were assessed using quantitative image analysis. RESULTS: At early incubation, methicillin-sensitive and methicillin-resistant S. aureus and Staphylococcus epidermidis, and cephalosporin-sensitive and cephalosporin-resistant K. pneumoniae and Acinetobacter baumannii colonies were significantly smaller in the SO-coated than in the control media. The number of colony-forming units showed no significant differences between SO-coated and control media for all tested species. CONCLUSION: SO demonstrated an inhibitory effect on the early colony expansion of certain aerobic bacteria, including antibiotic-resistant bacterial strains, but does not have a bactericidal effect. Therefore, the use of SO tamponade should be primarily determined by the clinical presentation and intraoperative retinal findings during surgery, rather than its potential antimicrobial effects.