Aims and objectives: The aim of this study was to identify the organisms responsible and to determine the antibiogram of the bacterial isolates from conjunctiva, nasal mucosa and lacrimal sac in cases of chronic dacryocystitis. Design: Prospective longitudinal study. Methods: A prospective analysis of 50 lacrimal sac contents of patients with chronic dacryocystitis conducted during the period from June 2019 to May 2020. Material was obtained directly from the lacrimal sac while making sac flap during external dacryocystorhinostomy for chronic dacryocystitis. Samples were also collected from ipsilateral conjunctiva and inferior meatus of nose. The specimens were cultured and results analyzed. Statistical Analysis: The results were analyzed by using Chisquare (χ2 ) test. Results: All the patients were in age group of 30 and above. Gram positive bacteria i.e., Coagulase negative staphylococci and Staphylococcus aureus were the most common isolates (60% and 25% respectively). Vancomycin, Linezolid, 3rd generation cephalosporins and amoxyclav were most sensitive antibiotics (82%, 85%, 81%, and 80% respectively). Comparison between lacrimal and nasal/conjunctival isolates showed high degree of resemblance between the isolates of two sites (p< 0.05). Conclusion: Majority of the chronic dacryocystitis cases are caused by Staphylococcus aureus and Coagulase negative Staphylococci. Amoxyclav and 3rd generation cephalosporins can be used to treat chronic dacryocystitis. Commensal flora of nose and conjunctiva shown a direct role in pathogenesis of chronic dacryocystitis.