Purpose To evaluate bacterial contamination of conjunctiva and aqueous humor in dogs undergoing phacoemulsification following asepsis with 0.5% povidone iodine and determine the influence of intravenous antibiotics on outcome of contamination. Methods Client‐owned dogs were prospectively enrolled and randomly assigned to a control group, receiving 22 mg/kg intravenous cefazolin at induction prior to sampling, or experimental group receiving no antibiotic prior to sampling, masked to the surgeon. Dogs receiving antimicrobials in the pre‐operative period were excluded. Asepsis was performed on all operated eyes using 0.5% iodine with minimum 3 min contact time at induction of anesthesia and repeated before surgery. A conjunctival swab and aqueous humor sample were collected prior to incision and following incision closure, respectively. Samples were submitted for aerobic and anaerobic bacterial culture and susceptibility. Results Seventy‐one eyes of 42 dogs were included. Median age was 9 years. Thirty‐nine and 32/71 eyes received intravenous cefazolin and no antibiotic, respectively. Median procedure time was 40 min per eye. Conjunctival cultures were positive in 6 eyes (8.5%): Serratia marcescens (5 eyes) and Cutibacterium acnes (1 eye). Aqueous humor cultures were positive in 5 eyes (7.0%): S. marcescens (2 eyes), Pseudomonas aeruginosa (2 eyes), Staphylococcus pseudointermedius (1 eye). Prevalence of positive culture did not differ between groups (p = .74), order of eyes for bilateral procedures (p = .74) and diabetic status (p = 1). Conclusions Bacterial contamination of the conjunctiva and aqueous humor was present in 8.5% and 7.0% of dogs undergoing phacoemulsification after asepsis. Lack of IV cefazolin was not significantly associated with positive culture.