In simulations of Ebola-level personal protective equipment doffing with experienced healthcare workers, hands, inner gloves, and scrubs are contaminated with nonenveloped viruses and, infrequently, with enveloped viruses.Personal protective equipment (PPE) protects healthcare workers (HCWs) caring for patients with Ebola virus disease (EVD), and PPE doffing is a critical point for preventing viral self-contamination. We assessed contamination of skin, gloves, and scrubs after doffing Ebola-level PPE contaminated with surrogate viruses: bacteriophages MS2 and I broken vertical bar 6. In a medical biocontainment unit, HCWs (n = 10) experienced in EVD care donned and doffed PPE following unit protocols that incorporate trained observer guidance and alcohol-based hand rub (ABHR). A mixture of I broken vertical bar 6 (enveloped), MS2 (nonenveloped), and fluorescent marker was applied to 4 PPE sites, approximating body fluid viral load (I broken vertical bar 6, 10(5); MS2, 10(6)). They performed a patient care task, then doffed. Inner gloves, face, hands, and scrubs were sampled for virus, as were environmental sites with visible fluorescent marker. Among 10 HCWs there was no I broken vertical bar 6 transfer to inner gloves, hands, or face; 1 participant had I broken vertical bar 6 on scrubs at low levels (1.4 x 10(2)). MS2 transfer (range, 10(1)-10(6)) was observed to scrubs (n = 2), hands (n = 1), and inner gloves (n = 7), where it was highest. Most (n = 8) had only 1 positive site. Environmental samples with visible fluorescent marker (n = 21) were negative. Among experienced HCWs, structured, observed doffing using ABHR protected against hand contamination with enveloped virus. Nonenveloped virus was infrequent on hands and scrubs but common on inner gloves, suggesting that inner gloves, but not necessarily ABHR, protect against hand contamination. Optimizing doffing protocols to protect against all types of viruses may require reinforcing careful handling of scrubs and good glove/hand hygiene with effective agents.