This paper aims to assess the antiseptic capacity of a double-distilled ozonated water solution and compare it to standard presurgical antiseptic protocols based on chlorhexidine and alcohol. Sixty-three canine patients undergoing different surgical procedures (such as thoracic, abdominal, proximal limb, or cervical) were enrolled in the study. Following extensive preoperative clipping of the selected anatomical regions, four standardized skin areas (6 × 6 cm each) were aseptically sampled according to a predefined protocol: Area 1 received soap-based chlorhexidine followed by alcoholic chlorhexidine; Area 2 was prepared using a combination of soap-based chlorhexidine and ozonated water; Areas 3 and 4 underwent presurgical skin antisepsis with a combination of ethyl alcohol and ozonated water, and ozonated water alone, respectively. Two different samples per area were collected using bacteriological buffers in order to evaluate the bacterial count at 90 s and 180 s, respectively. Those samples were sown via streak plating on Petri dishes using PCA (plate count agar) as the medium. The determination of the total mesophilic bacterial count on Petri dishes was used to assess the efficacy of the antiseptics in different areas. Results show that ozonated bidistilled water demonstrated comparable or superior efficacy compared to other antiseptics tested in this study. Its integration into surgical protocols could enhance patient safety while controlling environmental concerns associated with the massive use of chemical antiseptics.