BACKGROUND: To evaluate the antimicrobial activity of lidocaine (LD) topical anesthetic spray against oral microflora. METHODS: Antimicrobial effects of 10% LD spray were assessed against six bacterial cultures obtained from volunteers: Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Streptococcus salivarius, Streptococcus pyogenes, and Streptococcus sanguinis. The filter papers contained 50-µl LD, brain heart infusion (BHI) broth, or 0.2% chlorhexidine. Papers were placed on the cultured blood plates for 1-3 min. After the papers were removed, plates were incubated for 24 h. Bacterial growth on the contact areas was recorded as the antimicrobial score. The split mouth technique was use in for sample collection in clinical study. Filter papers soaked with either BHI broth or LD were placed on the right or left buccal mucosa for 1 min, and replaced with other papers to imprint biofilms onto the contact areas. Papers were placed on blood plates, incubated for 24 h, and antimicrobial scores were determined. Experiments were conducted for 2- and 3-min exposure times with a 1-day washout period. RESULTS: LD exhibited bactericidal effects against E. coli, S. sanguinis, and S. salivarius within 1 min but displayed no effect against S. aureus, E. faecalis, and S. pyogenes. The antimicrobial effect of LD on oral microflora depended upon exposure time, similar to the results obtained from the clinical study (P < 0.05). LD showed 60-95% biofilm reduction on buccal mucosa. CONCLUSIONS: Antimicrobial activity of 10% LD topical anesthetic spray was increased by exposure time. The 3 min application reduced oral microflora in the buccal mucosa.