Background: The conjunctiva of the eye is a mucosal surface that is colonized by various bacteria. Following surgeries, trauma, or other underlying conditions the normal flora of these bacteria may cause some eye infections such as conjunctival, corneal infections, or endophthalmitis. Objectives: This study aimed to investigate the bacterial population and antibiotic resistance of the conjunctiva in healthy horses. Study design: Random sampling of ocular samples from 20 horse during the winter (2019). Methods: Swab samples were collected from the eyes of 20 healthy horses of different breeds, ages, and sexes in Tehran province, Iran. The swabs were cultured on blood agar and MacConkey agar and incubated at 37°C for 24-48 hours. The isolated bacteria were characterized using cellular morphology, gram-staining, and biochemical tests. Sensitivity and resistance to 10 different antibiotics were measured using an antibiogram test. Results: Results showed that the bacterial population consisted of various species, with Bacillus cereus being the most frequent. There were no significant differences in the frequency of isolated bacteria between sexes and age groups. There were no significant differences in the sensitivity of bacterial flora of the eyes to Colistin, ceftiofur, florfenicol, amoxicillin, and ampicillin between neither different age groups nor different sexes of the horses (P>0.05). However, sensitivity to enrofloxacin and ciprofloxacin was significantly correlated with the age groups of the horses (P>0.05). Significant difference in sensitivity to trimethoprim-sulfamethoxazole between various sexes of horses was observed. Main limitations: The non-uniformity of the follow-up examination reliance owners or caregiver of the horse for follow-up information and Lack of adequate financial resources to study more horses. Conclusions: The study suggests that the frequency of bacterial flora in horses’ eyes is influenced by housing and management conditions rather than age and sex. Keywords: Microbial flora, Conjunctiva, Eyes, Horse, Mucosal surface.