Antibacterial effect of tetracaine hydrochloride was studied. Tetracaine hydrochloride (preservative free) were incubated with Staphylococcus aureus, coagulase negative staphylococcus and Pseudomonas aeruginosa respectively, for 18 hours and for 2 minutes. Then it was diluted and cultured on nutrient agar plate. Colony counts were done after 18 hours. In case of 18 hours incubation, there was no growth of microbials in 0.5%, 0.1% tetracaine hydrochloride, but there was no inhibitory effect of 0.02% tetracaine hydrochloride on growth of microbials, irrespective of inoculum amount. In case of 2 minutes incubation with 0.5% tetracaine hydrochloride, there was no difference between the amount of microbial inoculum and colony count. Above indicates that tetracaine hydrochloride has no inhibitory effect on bacterial growth after short exposure of less than 2 minutes.