Background: The objective of the study was the evaluation of the infection of ticks with pathogenic microorganisms at various workplaces (timber acquisition, forest growing, forest cultivation, forest protection). Material and Methods: Eight hundred sixty one Ixodes ricinus ticks collected from 4 workplaces were examined for the presence of Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti by polymerase chain reaction (PCR). Then, a comparative analysis of the relative density and infection of ticks at individual workplaces was done. In the statistical analysis, Chi² test, and Pearson’s test for correlation were applied. Results: The differences in infection (15.9–50%) of ticks with B. burgdorferi between the examined workplaces were highly significant, with the highest percentage observed at forest growing. The percentages of infection of ticks with A. phagocytophilum at individual workplaces ranged from 1.1–3.7%, and differences were statistically insignificant. The percentages of infections of ticks with Babesia microti at individual workplaces fluctuated from 3.6–4.4% and differences were also insignificant. Co-infections of ticks with 2 or 3 pathogens were rare. Conclusions: Co-infections with B. burgdorferi and B. microti showed a significant relationship with the workplaces, while those with B. burgdorferi and A. phagocytophilum did not show such a dependence. No significant positive correlation was found between the relative density of ticks and the frequencies of infections with B. burgdorferi, A. phagocytophilum and B. microti. Med. Pr. 2014;65(5):575–581