Aims: To investigate the microbiology of specimens from patients with chronic dacryocystitis. Methods: A total of 100 samples were obtained from the contents of the lacrimal duct in 91 consecutive patients with chronic dacryocystitis, including nine bilateral cases. These samples were cultured for aerobic and anaerobic bacteria and fungi with a routine microbiological technique. Positive cultured isolates were identified. Results: A total of 180 strains were isolated from the 85 bacterial samples, including 150 strains of aerobic bacteria and 30 strains of anaerobic bacteria. Fungi were found in eight samples and actinomyces in three samples. The bacterial species that were most frequently found were Staphylococcus species, representing 34.5% of all strains, followed by Corynebacterium diphtheroides (15.5%). The sensitivity tests revealed that levo-ofloxacin and amikacin were the most effective antibiotics. Conclusions: Gram positive cocci was the predominant pathogen of chronic dacryocystitis. Levo-ofloxacin and amikacin had the best effect in vitro.