Fluoroquinolones, including second generation (ofloxacin and ciprofloxacin), third generation (levofloxacin and tosufloxacin) and fourth generation (gatifloxacin and moxifloxacin), are a group of bactericidal agents used to treat multiple ocular infections. The third and fourth generation fluoroquinolones have enhanced Gram-positive activity when compared to the earlier ones. However, long-term use of these can affect human corneal epithelial cells. This study looks at the biologic and toxic effects of these diverse generation fluoroquinolones on cultured human corneal epithelial cells. Amongst those studied were ofloxacin, levofloxacin, tosufloxacin, moxifloxacin and gatifloxacin. MTT-based calorimetric assay, lactate dehydrogenase (LDH) assay and scratch wound test were performed. Corneal epithelial cell morphologies were examined by performing inverted light microscopy and transmission electron microscopy. The study found that the fourth generation fluoroquinolones (moxifloxacin and gatifloxacin) induced greater toxic effect to the corneal epithelial cells when compared to previous generation fluoroquinolones.