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Microbiological contamination of the anesthesia work environment (AWE) is a potential source of health care–associated infections. Medication syringes, stopcocks, and many other areas are routinely contaminated during anesthetic care, and adherence to hand hygiene recommendations is poor. Using a simulation model, we investigated whether AWE contamination could be reduced by implementing an intervention bundle focused on infection prevention.Twenty-five anesthesia providers were enrolled in this nonrandomized simulation scenario crossover design study. Subjects were asked to complete 2 general anesthesia scenarios in a mock operating room: a baseline scenario and an intervention scenario in which the bundle was implemented. The bundle included: double gloving before intubation, confining all airway equipment to 1 area, and performing hand hygiene before touching the anesthesia cart. Before each scenario, a manikin’s oropharynx and face were marked with ultraviolet fluorescent tracers. After each scenario, the AWE was inspected with a ultraviolet light source to detect contaminant, and all sites were photographed. A blinded observer scored the images for the presence or absence of tracer at 20 sites. Videos of the scenarios were analyzed for duration and number of hand hygiene and glove removal events. Data were analyzed using a mixed effects model. Subjects completed a survey about their experience and the value of the scenarios.The intervention was associated with a decreased subject contamination score of 4.0 (95% confidence interval, 2.2–5.6; P < .001), a 27% reduction in score between baseline and intervention scenarios. Some sites were universally contaminated despite the intervention (eg, laryngoscope handles). The intervention had a statistically significant impact on reducing contamination on medication syringes and the anesthesia cart. There was no significant difference in time needed to complete baseline and intervention scenarios. The majority of subjects felt that the simulations had significant value and would affect their future clinical behavior.Our results support the concept of an infection prevention bundle in reducing AWE contamination. Anesthesia providers deliver care in a unique environment in which “clean” and “contaminated” tasks are performed rapidly and often in parallel. Linking hand hygiene to specific high-impact tasks such as administering medications, designating areas for contaminated equipment, and double gloving before airway management are simple steps that can be implemented rapidly and are compatible with timely patient care. Our study has improved awareness of infection prevention issues in our department, and has highlighted simple and achievable actions that have the potential to reduce health care–associated infections.