Objective To determine the incidence of perforation of surgical gloves and identify associated risk factors that contribute to glove perforation in small animal ophthalmic surgery. Study design Observational cohort study. Sample population Surgical gloves (n = 2000) collected following 765 small animal ophthalmic procedures. Methods All the gloves were tested for perforation at the end of the procedure using a water leak test. The potential risk factors for glove perforation were recorded, and associations between these risk factors and perforation were explored using univariable (Fisher's exact test) and mixed effect logistic regression analysis. Results were considered significant if P < .05. Results Glove perforation was detected in 6% of procedures. Glove perforation was 1.97 (95% CI: 0.98‐4.22) times more likely in extraocular than in intraocular surgeries (7.3% vs 3.9%; P = .0462). The incidence of perforations was not statistically different between main and assistant surgeon (P = .86). No significant association was found between the risk of glove perforation and duration of the procedure (P = .13). Perforation of the nondominant hand was 2.6 (95% CI: 1.38‐4.98) times more likely than the dominant hand (74% vs 26%; P = .0028). Only 22% of the perforations were detected intraoperatively. Multivariable analysis identified only extraocular surgery as a risk factor for perforations. Conclusions There is a low incidence of glove perforation in small animal ophthalmic surgery, but extra care of the nondominant hand is required, especially during extraocular procedures.