Hand hygiene is the most effective way to prevent nosocomial infections. Nevertheless, the hands of healthcare professionals are still the primary route of transmission of pathogens responsible for such infections. The aim of this study was to evaluate hand disinfection techniques and investigate the risk factors that may explain the improper hand disinfection techniques among healthcare workers. We selected 7544 hospital workers directly involved in patient care. We recorded data based on the questionnaires, demographic data, and the preparation of hands for disinfection, including risk factors. Correct hand disinfection was verified by COUCOU BOX, with a UV camera. Proper hand disinfection was demonstrated among 4879 (64.7%) subjects, while 2665 (35.3%) subjects disinfected their hands incorrectly. In most places of work, nurses properly disinfected their hands more often than the physicians, particularly in general departments (62.1% vs. 69.2%; p = 0.0019). We observed that long nails and artificial/polished nails were more often observed in the group of nurses than in the group of physicians (7.3% vs. 4.7%, respectively; p = 0.0006 and 19.3% vs. 10.1%; p = 0.0000), while an inverse relationship was found in relation to watches (24.0% vs. 12.0%; p = 0.0000) and long sleeves (24.4% vs. 8.1%; p = 0.0000). Incorrect and less effective hand hygiene among some groups of hospital workers is still present. Therefore, the continuation of education actions concerned with hand hygiene among healthcare workers is needed.