The aim of this study was to investigate the effect of topically applied ocular anesthetic proparacaine on conjunctival and nasal bacterial mucosal flora in patients with dry eye disease. A Schirmer test was done with (group 1) and without (group 2) topical anesthetic proparacaine to 40 patients in each group. Conjunctival and nasal cultures were obtained before and 10 min after performing the Schirmer test. The bacterial culture results and the isolated bacteria were recorded in two groups. Patients' mean age was 62 years (70 female, 10 male). Before the application of topical anesthetic, 50 (62.5%) and 62 (77.5%) had positive conjunctival and nasal culture, respectively, with the most commonly isolated organism being coagulase-negative Staphylococcus in each group. In group 1 the conjunctival bacterial culture positivity rate decreased from 26 (65%) to six (15%) eyes (p < 0.001); however, this rate decreased slightly from 24 (60%) to 20 (50%) eyes in group 2 (p > 0.05). For the nasal cultures, the bacterial culture positivity rate decreased from 80% to 20% and from 75% to 65% in groups 1 (p < 0.001) and 2 (p > 0.05), respectively. Topical ocular anesthetic proparacaine has antibacterial activity in both conjunctival and nasal flora in patients with dry eye disease.