Purpose: To compare the in vitro activity of gatifloxacin with that of other topical antibiotics against fresh bacterial isolates from various types of human ocular infections. Methods: A total of 498 strains of ocular bacterial isolates were tested for their susceptibility to gatifloxacin, ofloxacin, ciprofloxacin, norfloxacin, gentamicin, tobramycin, neomycin, chloramphenicol, erythromycin, tetracycline and amikacin. Results: Ninety-nine percent of gram-positive and 92% of gram-negative bacterial strains were found to be susceptible in vitro to gatifloxacin, whereas lower percentages of gram-positive and gram-negative bacteria were found to be susceptible to other antibiotics. Conclusions: In vitro, gatifloxacin exhibits good activity against various bacterial species isolated from different ocular sites. This requires corroboration in the clinical setting.