OBJECTIVE. To determine the effectiveness of an automated ultraviolet-C (UV-C) emitter against vancomycin-resistant enterococci (VRE), Clostridium difficile, and Acinetobacter spp. in patient rooms. DESIGN. Prospective cohort study. SETTING. Two tertiary care hospitals. PARTICIPANTS. Convenience sample of 39 patient rooms from which a patient infected or colonized with 1 of the 3 targeted pathogens had been discharged. INTERVENTION. Environmental sites were cultured before and after use of an automated UV-C-emitting device in targeted rooms but before standard terminal room disinfection by environmental services. RESULTS. In total, 142 samples were obtained from 27 rooms of patients who were colonized or infected with VRE, 77 samples were obtained from 10 rooms of patients with C. difficile infection, and 10 samples were obtained from 2 rooms of patients with infections due to Acinetobacter. Use of an automated UV-C-emitting device led to a significant decrease in the total number of colony-forming units (CFUs) of any type of organism (1.07 log10 reduction; P < .0001), CFUs of target pathogens (1.35 log10 reduction; P < .0001), VRE CFUs (1.68 log10 reduction; P < .0001), and C. difficile CFUs (1.16 log10 reduction; P < .0001). CFUs of Acinetobacter also decreased (1.71 log10 reduction), but the trend was not statistically significant (P = .25). CFUs were reduced at all 9 of the environmental sites tested. Reductions similarly occurred in direct and indirect line of sight. CONCLUSIONS. Our data confirm that automated UV-C-emitting devices can decrease the bioburden of important pathogens in real-world settings such as hospital rooms.