Niepołomice Forest is located about 20 kilometers east of Cracow (Malopolska province, southern Poland). Its natural and touristic values, as well as wide range of hosts occurring within indicate this to be an area of high risk of exposure to Ixodes ricinus and tick-borne diseases it transfers. I. ricinus is a common species in Poland and Europe. Its seasonal activity begins in Poland in the early spring, and ends with late autumn. A total number of 129 specimens of I. ricinus was collected by flagging in Niepołomice Forest. DNA was isolated by ammonia method from 30 randomly-selected individuals. PCR was used to detect tick-borne pathogens with primers specific for Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and Babesia sp. Molecular studies confirmed the presence of all three pathogens in I. ricinus. A. phagocytophilum was found in 76.7%, Babesia sp., 60%, B. burgdorferi s. l., in 3.3% of studied ticks. A. phagocytophilum co-infection with Babesia sp., was found in 46.7% of the specimens. A co-infection of all three tested pathogens was recorded in one case (3.3%). In Poland the problem of tick-borne diseases is a growing issue, therefore people residing in southern Polish touristic areas should be informed about the prevention and protection against ticks.