Objective To analyze the distribution of pathogenic bacteria and their drug resistance in patients with ocular infection in a tertiary hospital in Beijing, and to provide reference for the clinical rational use of antibiotics. Methods A retrospective analysis was conducted to statistically analyze the composition and drug resistance of specimens submitted by patients with ocular infection in a tertiary hospital in Beijing from January 2021 to December 2023. Results A total of 255 pathogenic strains were isolated, with a positive rate of 56.42%. Among them, 134 （52.55%） strains of Gram-positive bacilli, 71 （27.84%） strains of Gram-negative bacilli, 39 （15.29%） strains of Gram-positive bacilli, and 11 （4.31%） strains of fungi were detected. The top 5 pathogenic bacteria isolations were Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium, Pseudomonas aeruginosa, and Streptococcus viridans, accounting for 18.04%, 14.09%, 10.20%, 7.45%, and 7.06%, respectively. The results of drug susceptibility tests showed that Gram-positive cocci had the highest resistance rate to penicillin and erythromycin, and the lowest resistance rate to vancomycin and linezolid. Enterobacteriaceae had lower resistance rate to levofloxacin and ciprofloxacin, at 28.00% and 24.00%, respectively. The overall resistance rate of Pseudomonas aeruginosa was relatively low. Conclusions The isolated bacteria from patients with ocular infection are mainly staphylococci, with Staphylococcus aureus being the most common one. Gram-positive bacteria are generally resistant to penicillin and erythromycin, but have lower resistance rate to rifampicin,and can be a better choice in clinically practice. The resistance rate of Gram-negative bacteria is generally low, and quinolones and aminoglycosides can be a choice in empirical practice.