Background: Knowledge of occupational health is crucial to the safety of healthcare workers in the pandemic period. The aim of our study was the rating of SARS-CoV-2 seroprevalence in connection with selected demographic, social, and organizational factors, as well as the identification of key elements determining the safety of HCWs and patients of the University Hospital in Krakow. Methods: This was a non-interventional, uncontrolled, open, single-center, cross-sectional online survey on the preparedness for the COVID-19 epidemic and the seroprevalence of medical and non-medical HCWs and students. Serum specimens from 1221 persons were tested using an immunoassay analyzer based on the ECLIA technique for the anti-SARS-CoV-2 antibodies IgM + IgG. Results: The total seroprevalence was 42.7%. In medical students it was 25.2%, while in physicians it was 43.4% and in nurses/midwives it was 48.1%. Of those who tested positive, 21.5% did not know their serological status. The use of personal protective equipment did not have any significant impact on the result of testing for anti-SARS-CoV-2 antibodies. The risk of developing the disease was not influenced by sex, professional work experience, workplace, or intensity of contact with the patient. Among the studied elements, only care of COVID-19 patients significantly increased the risk. The protective factor was starting work between the waves of the epidemic (June–September 2020). Conclusions: PPE is only one element of infection prevention and control—without other components, such as hand hygiene, it can be dangerous and contribute to self-infection. It is also very important to test healthcare workers. Not being aware of the COVID-19 status of HCWs poses a threat to other staff members, as well as patients and the family and friends of the infected. Thus, extreme caution should be applied when employing respirators with exhalation valves during the COVID-19 pandemic.