This retrospective observational study describes the results of an ad-hoc designated prevention protocol aimed at containing the spread of the scabies infestation among healthcare workers (HCWs) of a large University Hospital in Italy. The outbreak started on October 2022 and a preventive protocol was set up thanks to a multidisciplinary approach. HCWs at high scabies risk were defined as subjects working in Operative Units with a scabies prevalence higher than 2%, close contacts of a confirmed case of scabies, or HCWs with signs and symptoms of the disease. All cases at high scabies risk underwent a dermatological examination, and the infested HCWs were suspended from work until definitive healing. Mass drug administration was established for all HCWs working in Operative Units with a scabies prevalence higher than 2%. Until March 2023, out of 183 screening dermatological examinations, 21 (11.5%) were diagnostic for scabies. Between 11 October 2022 (date of the first diagnosed scabies case) and 6 March 2023 (the end of incubation period related to the last case detected), the frequency of scabies was 0.35% (21 scabies cases/6000 HCWs). The duration of the outbreak in our hospital was 14.7 weeks. Statistical analysis shows a significant association between scabies and being a nurse and having an allergy to dust mites. We obtained a low frequency of scabies infection, limiting the duration of the outbreak and the related economic burden.