Background: Correct reprocessing and microbiological surveillance on endoscopes are fundamental for preventing the transmission of multi-drug resistant strains and device-related infections. Methods: A questionnaire with three domains was created: (1) centre characteristics; (2) endoscope reprocessing procedures; and (3) application of microbiological surveillance. Nurses working in endoscopic units across Italy were invited to anonymously fill out the questionnaire on the SurveyMonkey platform between November 2021 and February 2022. Results: A total of 82 out of 132 endoscopic centres participated in the survey, with at least one centre from each Italian region. Data found different concerns regarding the current practice of both reprocessing and microbiological surveillance. According to respondents, the training on reprocessing was performed through theoretical training and only in 10% of centres; the microbiological surveillance was regularly performed in 59% of centres; and sampled endoscopes were not excluded for use in 31% of centres performing the surveillance until the outcome was pending, and when positive, 72% maintained them in quarantine until a successive negative result. Conclusions: Reprocessing and microbiological surveillance currently present several criticisms along the endoscopic centres in Italy. Our survey highlights the need for the correct application of the national recommendations in each endoscopic centre to prevent the potential transmission of endoscope-related infections.