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Objective: To identify ways that the built environment may support or disrupt safe doffing of personal protective equipment (PPE) in biocontainment units (BCU)., Design: We observed interactions between healthcare workers (HCWs) and the built environment during 41 simulated PPE donning and doffing exercises., Setting: The BCUs of 4 Ebola treatment facilities and 1 high-fidelity BCU mockup.ParticipantsA total of 64 HCWs (41 doffing HCWs and 15 trained observers) participated in this study., Results: In each facility, we observed how the physical environment influences risky behaviors by the HCW. The environmental design impeded communication between trained observers (TOs) and HCWs because of limited window size or visual obstructions with louvers, which allowed unobserved errors. The size and configuration of the doffing area impacted HCW adherence to protocol, and lack of clear demarcation of zones resulted in HCWs inadvertently leaving the doffing area and stepping back into the contaminated areas. Lack of standard location for items resulted in equipment and supplies frequently shifting positions. Finally, different solutions for maintaining balance while removing shoe covers (ie, chair, hand grips, and step stool) had variable success. We identified the 5 key requirements that doffing areas must achieve to support safe doffing of PPE, and we developed a matrix of proposed design strategies that can be implemented to meet those requirements., Conclusions: Simple, low-cost environmental design interventions can provide structure to support and improve HCW safety in BCUs. These interventions should be implemented in both current and future BCUs.