Purpose.To evaluate and compare the in vitro antimicrobial activity of levofloxacin versus ciprofloxacin and ofloxacin against ocular isolates from patients with bacterial conjunctivitis.Methods.The in vitro antimicrobial susceptibilities of ocular isolates to levofloxacin, ofloxacin, and ciprofloxacin were determined using both the agar disk diffusion and broth dilution methods.Results.Disk diffusion susceptibility testing disclosed that 99% (100 of 101 isolates) of Gram-negative isolates and 98% (127 of 129 isolates) of Gram-positive isolates were susceptible to levofloxacin; 96% (97 of 101 isolates) of Gram-negative isolates and 78% (100 of 129 isolates) of Gram-positive isolates were susceptible to ofloxacin; and 94% (95 of 101 isolates) of Gram-negative isolates and 61% (79 of 129 isolates) of Gram-positive isolates were susceptible to ciprofloxacin. Broth dilution testing disclosed that 99% (72 of 73 isolates) of Gram-negative isolates and 98% (111 of 113 isolates) of Gram-positive isolates were susceptible to levofloxacin; 96% (70 of 73 isolates) of Gram-negative isolates and 92% (104 of 113 isolates) of Gram-positive isolates were susceptible to ofloxacin; and 95% (69 of 73 isolates) of Gram-negative isolates and 82% (93 of 113 isolates) of Gram-positive isolates were susceptible to ciprofloxacin.Conclusions.In this study, levofloxacin demonstrated superior in vitro activity against human bacterial conjunctival isolates compared with either ofloxacin or ciprofloxacin (levofloxacin > ofloxacin > ciprofloxacin).