Antibiotic pretreatment is routine for chronic dacryocystitis (DC) patients. Herein, the longitudinal effects of antibiotic pretreatment before dacryocystorhinostomy for DC patients were evaluated. Conjunctival and nasal swabs were collected longitudinally from 33 DC patients with and without antibiotic pretreatment, both before dacryocystorhinostomy and at 1, 2, and 4 weeks postdacryocystorhinostomy. Additionally, conjunctival sac swabs were collected from 46 healthy volunteers and 14 other ocular diseases patients. Comparisons focused on ocular/nasal microbiota and recovery outcomes. Compared to healthy participants, DC patients without antibiotic pretreatment exhibited greater ocular microbiota diversity before dacryocystorhinostomy. Although clinical recovery rates were comparable, our results suggest that, after antibiotic pretreatment, the ocular microbiota richness and diversity, and the composition alteration tendency, significantly changed 4 weeks after surgery. This implies that the ocular microbiota was more disturbed in patients who underwent antibiotic pretreatment compared to those without such treatment. Furthermore, two types of ocular microbiota and three types of nasal microbiota were identified in ocular diseases. This study provides comprehensive data on the ocular and nasal microbiota in DC patients with and without antibiotic pretreatment, along with other ocular diseases. This finding suggested that antibiotic pretreatment may not be necessary before dacryocystorhinostomy for DC patients, especially for nonsevere cases.