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To investigate the antimicrobial activity of silicone oil against endophthalmitiscausing agents in vitro.The antimicrobial activity of silicone oil was tested on Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida albicans, and Aspergillus spp. The bacteria and fungi were separately inoculated into 1,300 centistokes silicone oil. Control inoculations were done in two different media: physiologic saline and brain-heart infusion (BHI) for bacteria and Sabouraud broth and physiologic saline for fungi. From each medium, 0.001-mL samples were taken and plated in Petri dishes. After overnight incubation, colony-forming units (CFUs) were enumerated. Culturing from the initially prepared specimens, incubating overnight, and counting CFUs was repeated until no growth of microorganisms was seen in the silicone oil-containing media. Macroscopic photography of the colonies and light microscopic photography of microorganisms were performed.All the microorganisms showed an apparent decrease in CFUs, with elimination between 7 and 21 days in silicone oil. Colony-forming units of microorganisms remained stable in physiologic saline during the study, with the exception of gradual decrease in CFUs of S. aureus and S. epidermidis from the beginning of the third day. In BHI and Sabouraud broth, both bacteria and fungi showed a growth pattern that was compatible with the growth curve of microorganisms.Silicone oil has an antimicrobial activity against S. aureus, S. epidermidis, P. aeruginosa, C. albicans, and Aspergillus spp., which are common endophthalmitiscausing agents.