Antiseptic flushing of the canine prepuce and its exclusion from the surgical field are recommended before abdominal surgery to reduce the risk of bacterial contamination. The authors cultured the preputial cavity of 60 dogs prior to and following flushing with 0.05% chlorhexidine diacetate, 1% povidone-iodine, or 0.9% saline control. Bacterial growth was evaluated using a semiquantitative method, and bacterial organisms were subsequently identified. There were no significant differences between povidone-iodine and the saline control in any of the variables assessed. Chlorhexidine resulted in a significant decrease in the proportion of positive postflush cultures compared with povidone-iodine. Although not significant, the difference in adverse reactions between povidone-iodine (25%) and chlorhexidine diacetate (5%) suggests clinical relevance. Based on the results of this study, a 2 min flush with 0.05% chlorhexidine diacetate is recommended for presurgical preparation of the preputial cavity.