Objectives: The aim was to compare the prevalence of acute infection and seropositivity of SARS-CoV-2 among healthcare workers (HCWs) and medical students. Material and Methods: A high-volume, single-center analysis was conducted in the period of July 1‒August 1, 2020, at the Semmelweis University. Naso- and oropharyngeal samples were collected for polymerase chain reaction (PCR), and blood samples for anti-SARS-CoV-2 IgG. A questionnaire was also administered about the infection symptoms and the obtained results were assessed by profession and site of care delivery. Results: From the total cohort (N = 7948), 4478 (56%) and 3470 (44%) were health professionals and medical students, respectively. They were mainly female (67%), and the mean age of HCWs and students was 40 and 25 years, respectively. By profession, physicians (1.5%) and other HCWs (1.8%) showed a comparable SARS-CoV-2 exposure. International students had the highest (2.1%), whereas Hungarian students had the lowest (0.6%) prevalence of seropositivity. The highest prevalence was detected among the staff of COVID-19 wards (12.1%). By PCR, medical students showed the lowest occurrence of active infection with a prevalence of 0.17%, while physicians and other HCWs had a higher prevalence (1.46% and 1.71%, respectively). By site of care delivery, positive test results were the most frequent at COVID-19 wards (3.8%). Conclusions: Physicians and other HCWs showed comparable SARS-CoV-2 seropositivity prevalence, approximately twice as high as in the general population of Budapest. Hungarian students had lower prevalence of seropositivity than this reference. High prevalence among international students suggests that they had imported the infection. The very high prevalence of documented exposure among staff members at COVID-19 wards urges for improving the safety measures. Int J Occup Med Environ Health. 2022;35(2):209–16