PurposeTo investigate bacterial contamination of indirect ophthalmoscopes and condensing lenses used in three UK veterinary referral centers, and the impact of an implemented cleaning protocol.MethodsBacteriology samples from 10 indirect ophthalmoscopes and 10 condensing lenses were taken at each center (n = 30 T0), before initiating one of three cleaning frequencies (every 2 weeks/once weekly/daily) for 28 days. The most contaminated indirect ophthalmoscope and condensing lens from each center were re‐sampled 30 min prior to (T1; n = 9) and 30 min after (T2; n = 9) the final clean. Sensitivity testing was completed using MIC.ResultsSeventy‐three isolates representing 15 different bacterial populations (genus/species) were cultured from 36 of 48 (75%) swabs tested. The most frequently cultured isolates were Staphylococcus spp. 30%, Micrococcus 22%, and Bacillus 14%. Pseudomonas aeruginosa, Pantoea, and Staphylococcus pseudintermedius demonstrated resistance to >50% of antibiotics against which they were tested.Eighty‐three percent of T0 samples (54 isolates across 11 species, median 2 isolates/swab), all T1 samples (15 isolates across 8 species, median 2 isolates/swab), and 22% of T2 samples (4 isolates across 4 species, median 0 isolates/swab) were contaminated. Head contact points were most contaminated irrespective of time point. A T1 sample was 57 times more likely (95% CI: 2.4–1376) to have a positive culture than a T2 sample (p = .01).ConclusionsBaseline contamination was high, representing a potential source of nosocomial infection in ophthalmic patients and handlers of diagnostic equipment. No center implemented a cleaning protocol prior to this study. Routine cleaning reduces bacterial contamination.