Background: The work presents the intentional use of harmful biological agents based on National Register of Biological Agents (Krajowego Rejestru Czynników Biologicznych – KRCB) collected by June 2023. Material and Methods: The KRCB is a central database gathering notifications of the intentional use of biological agents in risk groups 2–4 in the workplace in accordance with the regulation of the Minister of Health of December 11, 2020 amending the regulation regarding biological agents. Results: with the regulation of the Minister of Health of December 11, 2020 amending the regulation regarding biological agents. Results: Notifications were sent by 712 enterprises for: diagnostic – 62%, research – 31% and industrial purposes – 7%. Notifications were most often sent by hospital and non-hospital clinical and diagnostic laboratories (33% each group) and higher education and research units – 16%. In total, 7352 workers (88% women, 12% men) were exposed to biological agents intentionally used at work. Biological agents from 2 and 3 group risk were used by 98% and 16% enterprises, respectively. The most frequently were used bacteria Escherichia coli (with the exception of non-pathogenic strains) – 5972 (81%) exposed workers, Staphylococcus aureus – respectively 5760 (78%) and Pseudomonas aeruginosa – 5155 (70%). Among the total workers, nearly 25% were exposed to at least 1 biological agent carcinogenic to humans, including viruses (hepatitis B virus [HBV] – 1800 workers, 24%; hepatitis C virus [HCV] – respectively, 1767, 24%; human immunodeficiency virus-1 [HIV-1] – 1557, 21%; human gammaherpesvirus type 4 (Epstein-Barr virus) – 199, 3%; Papillomaviridae – 52, <1%; human gammaherpesvirus type 8 [HHV-8] – 16, <1%; human T-lymphotropic virus type 1 [HTLV- 1] – 38, <1%; bacteria Helicobacter pylori – 299, 4%; parasites Schistosoma haematobium – 9, <1%). Conclusions: Intentionally used biological agents most often pose a threat to workers of hospital and non-hospital clinical and diagnostic laboratories, but a higher health risk concerns workers of and higher education and research units. Med Pr Work Health Saf. 2023;74(6):501–11.