Preparing the skin of rodents for surgery often involves multiple applications of antiseptic agents. However, fewer applications may achieve the same antiseptic outcome. We evaluated the antimicrobial efficacy and effects on intraoperative body temperature of various surgical scrub agents, including novel waterless alcohol-based (WAB) options. Prior to ventral laparotomy, female C57BL/6 mice were treated with 0.9% saline (control); 70% ethanol; 10% povidone-iodine alternated with saline or 70% ethanol; 2% chlorhexidine digluconate alternated with saline or 70% ethanol; or 1 of 3 WAB products-commercial surgical scrub A, commercial surgical scrub B, or a common commercial hand sanitizer. Core temperatures were recorded, and aerobic culture swabs were collected from the surgical site at multiple time points. Intraoperative temperature trajectories for animals treated with scrub B, 10% povidone-iodine with saline, or hand sanitizer did not differ from saline (control). Temperature trajectories of mice treated with other scrub agents did differ significantly from saline. Bacteria were not detected at the operative site after 3 scrubs of 70% ethanol or 10% povidone-iodine alternated with ethanol, 2 scrubs of scrub A or B, 1 scrub of hand sanitizer, and both 1 and 3 scrubs of 2% chlorhexidine alternated with ethanol. Scrub B and 2% chlorhexidine-ethanol demonstrated prolonged antibacterial efficacy. Histology of corresponding haired skin sections revealed no differences in postoperative healing between groups, and no postoperative infections occurred. These results indicate that various novel WAB disinfectants, particularly scrub B (61% ethanol and 1% chlorhexidine gluconate), mitigate intraoperative temperature effects associated with several traditional agents and combinations. Furthermore, reduction of skin bacterial load without adverse effects on healing was seen with fewer than triplicate applications of most tested agents. Ultimately effective skin preparation can be achieved by using only 1 or 2 applications of scrub, thus rendering the triplicate skin-prep method unnecessary in laboratory mice.