Relevance. The healthcare personnel’s adherence to hand antisepsis is а pressing problem during any epidemic outbreak. In the context of high biological hazards, the possibility of performing proper antiseptic procedures is reduced while nosocomial pathogen transmission and healthcare-associated infection (HAI) rate growth are promoted. Aims. Studying the factors determining the healthcare workers’ adherence to hand antisepsis during a pandemic infection spread (as exemplified by COVID-19). Materials and methods. The study was conducted during the entire COVID-19 pandemic. To estimate the rate of adherence rate by the healthcare personnel to hand hygiene, we conducted an anonymous questionnaire survey of the staff in infectious disease hospitals (2021–2022) as well as assessed the viral and bacterial contamination rates of the personnel’s hands and the hospital environment items (2021–2023). Epidemiological (descriptive-evaluation), bacteriological (conventional culture, AMR determination using a VITEK2 analyzer), molecular biological (RT-PCR, Sanger sequencing) and statistical (a questionnaire survey, building machine learning models) methods were used in the study. Results. Based on the results of the survey, the main factors influencing the formation of attitudes towards antiseptics among the staff of medical organizations with medical and non-medical education were identified. It was found that the pandemic spread of infections negatively affects the level of contamination of the hands of infectious diseases hospital staff, which amounted to 35.3% of doctors, 28.8% of nurses, and 25% of hospital cleaners. The spectrum of isolated pathogens included viruses (SARS-CoV-2) and opportunistic microorganisms (Enterococcus faecalis, Enterococcus faecium, Pantoea agglomerans, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli), including those with a wide profile of antimicrobial resistance. Conclusion. During the COVID-19 epidemic, the rate of adherence to hand antisepsis in the infectious disease hospital was 82.3% in the staff with a medical background and 71.2% in the staff without one. The results confirm the significant role of staff hands in the in-hospital transmission of dangerous pathogens and indicate the need for systematic monitoring of the level of knowledge on antiseptics among all employees of a medical organization, regardless of specialization and level of education.