Free-roaming cats represent a potential reservoir of infectious diseases. The most common co-infections of free-roaming cats include mixed viral, bacterial, fungal, yeast and parasitic infections. This study focuses on the occurrence of Chlamydia spp. feline immunodeficiency virus (FIV), feline leukaemia virus (FeLV) and their co-infections. The diseases accompanied by immune suppression, such as FIV, create favourable conditions for the onset of other diseases and co-infections. The result of co-infection may be a higher susceptibility for other pathogens, as well as the occurrence of more severe clinical symptoms.The study involved 168 (113♀ and 55♂) free-roaming adult cats during the years 2021-2022. All cats belonged to Slovak citizens with permanent residence in the Slovak Republic. Blood samples and swabs (Invasive EUROTUBO® Collection sterile swab, Deltalab O8191 Rubí, Spain) from the conjunctival sac were taken from 168 cats to be later tested by PCR and ELISA methods. Statistical analysis was also performed.The overall prevalence of Chlamydia spp. was 17.26%, of FIV 15.48%, and 5.95% of FeLV. The most significant finding in our study was 3.57% co-infection of FIV and Chlamydia spp. in tested cats.The observed prevalence of Chlamydia spp. FIV and FeLV indicates that the presence of these pathogens in populations of free-roaming cats is endemic.