Toxoplasma gondii is considered one of the most successful parasites of humans and animals. The ingestion of viable cysts through the consumption of undercooked pork is recognized as a significant route of human infection with T. gondii. The aim of this study was to investigate the presence of viable parasite in tissues of free-range pigs from the Zasavica Special Nature Reserve. All pigs were of the Mangulica breed, raised in a traditional way. The serological screening was performed using a modified agglutination test (MAT). The isolation of viable T. gondii was attempted by a bioassay of pig heart tissue in mice, while the real-time polymerase chain reaction (qPCR) targeted at the 529 bp repetitive element of T. gondii was used to detect parasitic DNA in digested hearts. Specific antibodies were detected in 12 out of 18 pigs examined. The bioassay was performed for five MAT-positive and one MAT-negative pig, and a total of three isolates were obtained. qPCR was performed for all samples, including one MAT-negative sample that was not bioassayed. The presence of T. gondii DNA was confirmed in all hearts with a positive bioassay as well as in one originating from seropositive and one from seronegative pig whose hearts were not bioassayed. The successful isolation of viable cysts, presence of risk factors (such as older age at the time of slaughter) and increased contact with the environment, along with the great appreciation of Serbian consumers towards home-cured Mangulica’s meat, make this breed worthy of consideration as a potentially important reservoir of human infection.