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 UOs with more than five confirmed cases of scabies, close contacts of a confirmed case of scabies, or HCWs with signs and symptoms of the disease. All cases of at high scabies risk underwent a dermatological examination and the infested HCWs were suspended from work until definitive healing. Mass prophylactic treatment was established for all HCWs working in UOs with more than five confirmed cases of scabies. Until March 2023, out of 183 screening dermatological examinations, 21 (11.5%) were diagnostic for scabies. We obtain a prevalence of scabies of 2.6% (21/800) and a duration of the outbreak of 14.7 weeks. Statistical analysis shows a significant association between scabies and being a nurse and have an allergy to dust mites. We obtained a low prevalence of scabies infection, limiting the duration of the outbreak and the related economic burden.