Backgrounds Health care workers’ (HCWs) knowledge of and compliance with personal protective procedures is a key for patients’ and personnel safety. The aim of this study was to assess which factors are associated with higher self-evaluations of training on infection prevention and control (IPC) and higher self-assessment of IPC practices used by HCWs regarding COVID-19 in University Hospital in Krakow, Poland, in January 2021. Material and methods This was an online survey on the preparedness for COVID-19 epidemic of medical/non-medical staff and medical students. Questions included in the survey concerned participants’ socio-demographic characteristics, hospital staff involvement in the training, knowledge about the hand hygiene, and adherence to IPC measures. Knowledge and Performance Index (K&PI) based on selected questions was constructed for to reflect both subjective (self-evaluation) of preparedness and objective IPC knowledge and skills of HCWs participated in the IPC training. Results A total of 1412 health care workers, including 129 medical students, participated in the study. The largest group, 53.6%, was made up of nurses and paramedics. Age of respondents significantly correlated with knowledge of IPC and with K&PI. The mean age of workers with high K&PI was 42.39 ± 12.53, and among those with low, 39.71 ± 13.10, p < 0.001. 51% UHK workers participated in IPC training, but 11.3% of physicians, 28.8% of other HCWs, and 55.8% of students did not know the IPC standard precaution. Most participants, 72.3%, felt that they had received sufficient training; however, 45.8% of students declined this. There was no correlation between self-reported preparedness and the K&PI, indicating that self-reported preparedness was inadequate for knowledge and skills. Nurses and paramedics assessed their knowledge most accurately. Participants with low K&PI and high subjective evaluation constituted a substantial group in all categories. Students least often overestimated (23.8%) and most often (9.6%) underestimated their knowledge and skills. Conclusions Our study revealed inadequate IPC practice, especially as it refers to the training programme. We confirmed the urgent need of including theory and practice of IPC in curricula of health professions’ training in order to provide students with knowledge and skills necessary not only for future pandemic situations but also for everyday work.