Background: The work is a report on the intentional use of harmful biological agents in workplaces in Poland based on National Register of Biological Agents (Krajowy Rejestr Czynników Biologicznych – KRCB) as of June 2025. Material and Methods: The KRCB is a central database gathering notifications of the intentional use of biological agents in risk groups 2–4 in work processes in accordance with the Regulation of the Minister of Health of December 11, 2020, amending the regulation regarding biological agents. Results: Notifications were sent by 749 enterprises for: diagnostic – 59.3%, research – 32.4% and industrial purposes – 8.1%. Notifications were most often sent by microbial laboratories (N = 438, 58.5%), scientific-research institutions (N = 229, 30.6%) and industrial enterprises (N = 82, 11.0%). In total, 7795 workers (87.6% women, 12.4% men) were exposed to biological agents intentionally used at work. Biological agents from risk groups 2 and 3 were used by 97.6% and 15.9% enterprises, respectively. The most frequently were used bacteria Escherichia coli (with the exception of non-pathogenic strains) – 4823 exposed workers (61.9%), Staphylococcus aureus – respectively 4655 (59.7%) and Pseudomonas aeruginosa – 4188 (53.7%). National Register of Biological Agents gathered 9 notifications of intentionally using of the biological agent carcinogenic to humans, including viruses (hepatitis B virus [N = 2015 workers, 25.8%], hepatitis C virus [N = 1982, 25.4%], human immunodeficiency virus type 1 [N = 1796, 22.7%], Epstein-Barr virus [N = 196, 2.5%], human papilloma virus [N = 60, 0.8%], human herpes virus type 8 [N = 16, 0.2%], human T-lymphotropic virus type 1 [N = 38, 0.5%]), bacteria (Helicobacter pylori [N = 327, 4.2%]), parasite (Schistosoma haematobium [N = 9, 0.1%]). Conclusions: Intentionally used biological agents most often pose a threat to workers of microbial laboratories, but a higher health risk concerns workers of scientific-research institutions. Med Pr Work Health Saf. 2026;77(1)