Background: The paper presents the intentional use of biological agents for industrial, diagnostic and research purposes in Polish enterprises. Material and Methods: The National Register of Biological Agents (Krajowy Rejestr Czynników Biologicznych – KRCB) is an online database that collects the data on the intentional use of biological agents at work in Poland. Results: As of December 2013 there were 533 notifications in KRCB, mainly for diagnostic (73%), research (20%) and industrial purposes (7%). Mostly there were hospital diagnostic laboratories (37%), and other laboratories (35%), as well as higher education and research institutions (11%). In total, 4015 workers (91.7% of women, 8.3% of men) were exposed to biological agents. Agents classified in risk group 2 were used in 518 enterprises, and in risk group 3 in 107 enterprises. Of those agents the following bacteria were the most frequently used: Escherichia coli except for non-pathogenic strains (455 enterprises and 3314 exposed workers); Staphylococcus aureus (445 and 3270); and Pseudomonas aeruginosa (406 and 2969, respectively). In 66 enterprises there were used biological agents recognized by the International Agency for Research on Cancer (IARC) as carcinogens. They are viruses: Epstein-Barr (7 enterprises, 181 exposed workers); hepatitis B (16 and 257); hepatitis C virus (15 and 243); human immunodeficiency virus (8 and 107); human papillomaviruses (2 and 4); parasites: Clonorchis viverrini (1 and 2); Clonorchos sinensis (1 and 2); Schistosoma haematobium (1 and 2) and bacteria Helicobacter pylori (15 and 230, respectively). Conclusions: The National Register of Biological Agents at Work permits to evaluate the situation of occupational exposure to biological agents used intentionally in enterprises in Poland. Med Pr 2015;66(1):39–47