Objective: To investigate the antimicrobial activities of the drugs used as topical anesthetics. Material and Methods: Test drugs (oxybuprocaine 0.4%, proparacaine hydrochloride 0.5%, lidocaine 10%) were tested for Staphylococcus aureus (S. aureus) ATCC 29213, Streptococcus pneumoniae (S. pneumoniae)ATCC 27336, Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853, standard strains of Escherichia coli (E. coli) ATCC 25922 and Candida albicans (C. albicans) using modified microplate alamar blue method (MABA) in different concentrations according to Clinical and Laboratory Standards Institute (CLSI) criteria. Results: S. Pneumoniae proliferated in none of the dilutions of three topical anesthetics. S. aureus proliferated in all dilutions of oxybuprocaine and lidocaine and in no dilutions of proparacaine hydrochloride. P. Aeruginosa proliferated in 0.2% dilution of oxybuprocaine and 2.5% and 5% dilutions of lidocaine and in all dilutions of proparacaine hydrochloride. C. albicans proliferated in 0.05% and 0.025 % dilutions of oxybuprocaine, 1.25% and 0.625% dilutions of lidocaine, 0.125%, 0.0625% and 0.0312% dilutions of proparacaine hydrochloride. E. Coll proliferated in all dilutions of oxybuprocaine, 0.625% dilutions of lidocaine and 0.125%, 0.0625% and 0.0312% dilutions of proparacaine hydrochloride. Conclusio: Topical anesthetics that are used before sample collection in superficial ocular infections can cause false results in cultures by inhibating proliferations of microorganisms. Thus, it is important to know the effects of topical anesthetics and dilutions on bacterial proliferation.