The aim of this study was molecular detection of Toxoplasma gondii in 36 natural surface water bodies in Poland, including preliminary genotype identification and determination of co-occurrence of this parasite with other protozoa that have been detected in previous studies. The examined DNA samples were obtained before to detect Cryptosporidium, Giardia and free-living amoebae. Nested polymerase chain reaction (PCR) based on B1 gene and sequencing was performed for both confirmation of parasite presence in water and genotype identification. T. gondii DNA was detected in 19.4% (7/36) water bodies, while in the case of other studies, T. gondii prevalence ranged between 0% and over 56%. These differences may be caused by natural variations in T. gondii occurrence as well as different sample volumes and methods of sample processing or DNA isolation and detection. Two cases of double contamination were reported: T. gondii with Cryptosporidium parvum and T. gondii with potentially pathogenic Acanthamoeba T4 genotype, thus there is a possibility of mixed infection in humans after occasional contact with water. Obtained T. gondii strains were genetically identical or closely similar (99.8%) to RH strain representing genotype I, however, further examinations involving more loci will be conducted to identify the genotype.