The objective of this study was to evaluate the level of bacterial contamination of otoscope cones in veterinary private practice, and to determine the most effective method of disinfection. Fifty small animal practices participated in this study, which included a detailed survey regarding otoscope cleaning, storage and usage and quantitative culture of the cleaned and stored otoscope cones. Using sterile technique, two cones from each of the 50 hospitals were swabbed and submitted for quantitative culture. Contamination was present in 29% of the samples and the following organisms were isolated: Flavobacterium brevis (10%), Pseudomonas aeruginosa (6%), Pseudomonas alcaligenes (4%), Staphylococcus intermedius (4%), Corynebacterium spp. (2%), Bacillus spp. (1%), Enterococcus faecalis (1%) Malassezia spp. (1%). There was no statistically significant difference between storage type (dry versus stored in solution) and for the instrumentation used to clean the cones (brush, cotton-tipped applicator, both versus none). There was a statistically significant difference between the different cleaning solutions (P < 0.001) and between the storage solutions (P = 0.003). A single most effective cleaning solution was unable to be determined due to the large number of solutions utilized. Cetylcide G (Cetylite Industries, Inc., Pennsauken, NJ, USA) was the most effective of the three most commonly used storage solutions (Cetylcide G, Benz-all, and 2% Chlorhexidine gluconate) when used as directed (P < 0.001). The level of contamination had a positive association with the frequency of cone use and a negative association with the frequency of storage solution replacement.