The aim of the study was to assess the acceptance level of COVID-19 vaccination among healthcare workers (HCW) and the general population in Poland at the start of the national COVID-19 vaccination program from 18–31 December 2020. A cross-sectional anonymous survey was conducted in a group of 1976 people: 1042 health professionals and 934 non-medical professionals using an on-line questionnaire. The most skeptical about the COVID-19 vaccine were students of non-medical faculties, non-medical professions, and administrative–technical health service staff (26.2%, 38.7% and 41.2%, respectively). The most positive attitude to vaccination was reported by doctors, medical students and pharmacists (80.6%, 76.9% and 65.7%, respectively). Doctors (64.7%) and medical students (63.7%) most often declared confidence in vaccines compared to nurses (34.5%). Distrust about vaccine safety was declared by nurses (46.6%) and pharmacists (40.0%). HCW encouraged others to vaccinate more eagerly (65.8%) than non-medical professions (28.3%). Thus, a considerable proportion of HCW in Poland expressed concern about vaccines just prior to the beginning of the COVID-19 immunization program. The significant decrease in the willingness to vaccinate observed in Poland towards the end of 2021 must be considered in the light of the serious COVID-19 vaccination hesitancy in the Polish population.