Background: Among no-touch automated disinfection devices, ultraviolet-C (UV-C) radiation has been proven to be one of the most effective against a broad spectrum of Aim: To evaluate the antimicrobial efficacy of an experimental UV-C robotic platform, under controlled conditions and in a real hospital scenario, when used to implement the standard cleaning operating protocol (SOP). Methods: In vitro, following dose calibration tests, bactericidal and virucidal efficacy were tested in accordance with American Society for Testing and Materials International Standard E3135-18. In hospital settings, 12 high-touch surfaces were sampled after healthcare activity (dirty condition), after SOP alone, and after SOP + UV-C treatment, with a total of 180 samples. Findings: In vitro, <4 mJ/cm2 was required to remove Staphylococcus aureus and Pseudomonas aeruginosa completely, 194 mJ/cm2 was required to inactivate adenovirus HadV5 completely, and 38.8 mJ/cm2 was sufficient to inactivate coronavirus 229E completely. In the real hospital scenario, the mean UV-C dose emitted on the sampled surfaces was 29.31 mJ/cm2. A significant difference was found after SOP alone (P1/40.022) and after SOP + UV-C treatment (P1/40.007) compared with the dirty condition. The average percentage reduction in the total viable count (TVC) was 67% after SOP alone and 96% after SOP + UV-C treatment. Conclusions: Comparison of the tests conducted in vitro and in the real hospital scenario showed that the efficacy of the UV-C robot was reduced in the hospital setting, as a higher dose was needed to obtain a reduction in the TVC . (c) 2024 The Healthcare Infection Society. Published by Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.