BackgroundHealthcare-Acquired Infections are a major problem in the world and within the healthcare delivery system. An estimated 5–10% and around 25% of hospitalized patients have healthcare-acquired infections in developed and developing countries, respectively. Infection prevention and control programs have proven to be successful in lowering the incidence and spread of infections. Thus, this evaluation aims to evaluate the implementation fidelity of infection prevention practices at Debre Tabor comprehensive specialized hospital in Northwest Ethiopia.MethodsA facility-based cross-sectional design with a concurrent mixed method was used to evaluate the implementation fidelity of infection prevention practices. A total of 36 indicators were used to measure adherence, participant responsiveness, and facilitation strategy dimensions. A total of 423 clients were administered for an interview, an inventory checklist, a document review, 35 non-participatory observations, and 11 key informant interviews were conducted. A multivariable logistic regression analysis was used to identify factors significantly associated with the satisfaction of clients. The findings were presented using descriptions, tables, and graphs.ResultThe overall implementation fidelity of the infection prevention practices was 61.8%. The dimensions of adherence to infection prevention and control guidelines were 71.4%, participant responsiveness was 60.6%, and facilitation strategy was 48%. In multivariable analysis, ward admission and educational level had a p-value of below 0.05 and were significantly associated with the satisfaction of clients with infection prevention practices at the hospital. The major themes that emerged in qualitative data analysis were healthcare worker-related factors, management-related factors, and patient- and visitor-related factors.ConclusionThe evaluation result of this study concluded that the overall implementation fidelity of infection prevention practice was judged to be medium and needed improvement. It included dimensions of adherence and participant responsiveness that were rated as medium, as well as a facilitation strategy that was rated as low. Enablers and barriers were thematized into factors related to healthcare providers, management, institutions, and patient and visitor relations.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12879-023-08263-3.