AIM: To observe the shifting hierarchy of the conjunctival florae in the patients who employed a long-time topical fluoroquinolone and characterize the consequent variations of their antibiotic sensitivity and virulence. METHODS: A total of 143 eyes (143 patients) who suffered from the non-infectious corneal ulcer and topically used fluoroquinolone more than 2wk were enrolled as the fluoroquinolone eye. The untreated fellow eye was considered as the contralateral eye. Seventy-five healthy subjects were selected as the control. The culture positivity and strains of the isolated conjunctival florae were observed. Their antibiotic susceptibility and expression of the virulence-related genes were detected. RESULTS: Florae were recovered from 84.0%, 37.1%, and 57.3% of the conjunctival swabs in the control, fluoroquinolone eye, and contralateral eye, respectively. The most frequently isolated microorganisms were Staphylococcus epidermidis (34.9%) in the control, followed by Staphylococcus aureus (17.5%), Staphylococcus saprophyticus (14.3%), Micrococcus (9.5%), Propionibacterium acnes (7.9%). However, those orderly ranks shifted to Staphylococcus aureus (34.0%), Propionibacterium acnes (20.8%), Candida albicans (17.0%), Pseudomonas aeruginosa (9.4%) in the fluoroquinolone eye. A growing number of the fluoroquinolone-resistant florae survived in the fluoroquinolone eye, accompanied by an increased expression of the virulence-related genes. CONCLUSION: A long-time topical fluoroquinolone leads to a shifting hierarchy of the conjunctival florae, accompanied by the consequent variations of the antibiotic sensitivity and virulence.