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<b>Background: </b> To determine the antibacterial activity of newer fluoroquinolones and compare their activity between ciprofloxacin-susceptible and resistant bacterial isolates from patients with keratitis and endophthalmitis<i> .</i> <b> Materials and Methods:</b> The minimum inhibitory concentration (MIC) of ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin and moxifloxacin was determined for 123 bacterial isolates, using E test<i> .</i> Among the 123 isolates, 68 were gram-positive (<i> Staphylococcus</i> spp, <i> Streptococcus</i> spp, <i> Corynebacterium</i> spp, <i> Bacillus </i> spp.) and 55 were gram-negative (<i> Pseudomonas aeruginosa)</i> . The bacterial isolates were divided into three groups: susceptible / intermediate/ resistant to ciprofloxacin. The MIC values for various fluoroquinolones were compared between the three groups and between gram-positive and gram-negative bacteria. <b> Results:</b> For gram-positive isolates, median MICs of fourth generation fluoroquinolones were lower than second generation. The median MIC was lowest for gatifloxacin and moxifloxacin (0.094 mg/ml) in ciprofloxacin-susceptible isolates of gram-positive bacteria. For ciprofloxacin-susceptible gram-negative bacteria, the median MIC of ciprofloxacin (0.19 mg/ml) was significantly lower than ofloxacin, levofloxacin, gatifloxacin and moxifloxacin (1.5, 0.5, 0.5 and 2 mg/ml respectively). Ciprofloxacin-resistant isolates of gram-positive bacteria showed higher MIC of levofloxacin, moxifloxacin and gatifloxacin though they remained susceptible to them. None of the fluoroquinolones were effective against ciprofloxacin-resistant gram-negative bacteria. Overall, for gram-positive bacteria, median MICs of levofloxacin, moxifloxacin and gatifloxacin were below ciprofloxacin, the MIC of gatifloxacin and moxifloxacin was equal for gram-positive bacteria. <b> Conclusions:</b> Levofloxacin, gatifloxacin and moxifloxacin are statistically more effective against gram-positive bacteria, the latter two being equally effective. Ciprofloxacin remains the most effective fluoroquinolone against gram-negative bacteria.