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We have recently shown that intraoperative bacterial transmission to patient IV stopcock sets is associated with increased patient mortality. In this study, we hypothesized that bacterial contamination of anesthesia provider hands before patient contact is a risk factor for direct intraoperative bacterial transmission.Dartmouth–Hitchcock Medical Center is a tertiary care and level 1 trauma center with 400 inpatient beds and 28 operating suites. The first and second operative cases in each of 92 operating rooms were randomly selected for analysis. Eighty-two paired samples were analyzed. Ten pairs of cases were excluded because of broken or missing sampling protocol and lost samples. We identified cases of intraoperative bacterial transmission to the patient IV stopcock set and the anesthesia environment (adjustable pressure-limiting valve and agent dial) in each operating room pair by using a previously validated protocol. We then used biotype analysis to compare these transmitted organisms to those organisms isolated from the hands of anesthesia providers obtained before the start of each case. Provider-origin transmission was defined as potential pathogens isolated in the patient stopcock set or environment that had an identical biotype to the same organism isolated from hands of providers. We also assessed the efficacy of the current intraoperative cleaning protocol by evaluating isolated potential pathogens identified at the start of case 2. Poor intraoperative cleaning was defined as 1 or more potential pathogens found in the anesthesia environment at the start of case 2 that were not there at the beginning of case 1. We collected clinical and epidemiological data on all the cases to identify risk factors for contamination.One hundred sixty-four cases (82 case pairs) were studied. We identified intraoperative bacterial transmission to the IV stopcock set in 11.5 % (19/164) of cases, 47% (9/19) of which were of provider origin. We identified intraoperative bacterial transmission to the anesthesia environment in 89% (146/164) of cases, 12% (17/146) of which were of provider origin. The number of rooms that an attending anesthesiologist supervised simultaneously, the age of the patient, and patient discharge from the operating room to an intensive care unit were independent predictors of bacterial transmission events not directly linked to providers.The contaminated hands of anesthesia providers serve as a significant source of patient environmental and stopcock set contamination in the operating room. Additional sources of intraoperative bacterial transmission, including postoperative environmental cleaning practices, should be further studied.