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Purpose: To investigate the effect of long-term use of antiglaucomatous drops (AGD), and benzalkonium chloride (BAK) on eyelid and conjunctival flora. Materials and Methods: Forty eyes of 24 subjects who used AGD with a mean usage time of 24±17 (6-72) months, and 40 eyes of 34 subjects who had no glaucoma history as a control group were recruited. In this study, 8 different AGD were used, and all drops contained benzalkonium chloride (BAK) 0.05% (range, 0.02% to 0.075) as a preservative. Cultures of conjunctiva and eyelid samples were obtained by dacron swab, inoculated onto blood and chocolate agar, and incubated at 37°C in an atmosphere of 5-7% CO2 for 2 days. This study was planned as a case-control study. All bacterial isolates was identified, and isolated bacteria in both groups and their sensitivities to antibiotics were compared. Results: Diphtheroid rods were the most frequent isolated bacteria from the conjunctivas in the control group and in the group that used AGD, 23% (9/40) and 25% (10/40), respectively. Coagulase negative Staphylococcus was the most frequently isolated bacteria from the eyelids in the control group and in the group that used AGD, 93% (37/40) and 83% (33/40), respectively. The bacteria isolated from the group that had used AGD were more resistant to antibiotics than the bacteria isolated from the control group (p>0.05). Isolated bacteria were most susceptible to vancomycin, netilmicin and amikacin. There was no statistically significant difference between the groups in the proportion of conjunctival and eyelids' cultures that yielded positive (p>0.05). Conclusions: Isolated bacteria in the group that used AGD were more resistant to antibiotics in comparison with the control group. We conclude that long-term use of AGD containing BAK, which is also a disinfectant agent, may cause colonisation with more resistant bacteria on conjunctivas and eyelids. [ABSTRACT FROM AUTHOR]