Objective The objective of this study was to evaluate the incidence of nosocomial infection and the impact of cross-transmission of SARS-CoV-2 among inpatients at a tertiary care teaching hospital. Methods This was a retrospective cohort study involving inpatients admitted to a tertiary university hospital in the city of Sao Paulo, Brazil, between March 2020 and February 2021. Cases were identified on the basis of a positive reverse-transcription polymerase chain reaction result for SARS-CoV-2 and the review of electronic medical records. Nosocomial transmission was defined by applying the criteria established by the Brazilian National Health Regulatory Agency. Results We identified 2146 cases of SARS-CoV-2 infection, 185 (8.6%) of which were considered cases of nosocomial transmission. The mean age was 58.3 years. The incidence density was 1.78 cases per 1,000 patient-days on the general wards, being highest on the cardiac surgery ward, and only 0.16 per 1,000 patient-days on the COVID-19 wards. Of the 185 patients evaluated, 115 (62.2%) were men, 150 (81.1%) cases had at least one comorbidity, and 104 (56.2%) evolved to death. Conclusions Despite the preventive measures taken, nosocomial transmission of SARS-CoV-2 occurred throughout our hospital. Such measures should be intensified when the incidence of community transmission peaks.