We prospectively investigated contamination of high-contact surfaces in the operating room (OR) using adenosine triphosphate (ATP) monitoring. We tested whether contamination would increase from morning (AM) to afternoon (PM), despite cleaning between cases. Second, we compared the degree of OR contamination to non-OR control sites. ORs with high case volumes were selected for the study. Ten sites in each OR were swabbed using the AccuPoint® HC ATP Sanitation Monitoring device, which provided a numerical measure of contamination (relative light units, RLUs). According to the manufacturer, surfaces are considered clean at ≤ 400 RLUs. AM measurements were taken before the start of surgical cases and PM measurements were taken after cases were completed. Eighty morning and 70 afternoon samples were obtained from 8 ORs. Apart from the OR floor, laryngoscope handles had the highest level of morning contamination (1204 RLUs, interquartile range 345, 2603), with 75% of AM samples and 100% of PM samples exceeding 400 RLUs. This contamination was comparable to hospital toilet seats (87% of samples exceeding 400 RLUs). No sites showed statistically significant increases in contamination from AM to PM. Apart from the OR floors, laryngoscope handles emerged as a key OR site where improved cleaning practices may reduce cross-contamination risk. While some sites showed increased contamination over the course of the day, none of these met statistical significance thereby offering tentative evidence that current cleaning practices during case turnover are effective for most sites.