Context: The contamination of soil by pathogenic protozoan parasites from both human and animal feces, is an important factor in the spread of parasitic infections globally. This contamination also serves as a crucial reservoir for zoonotic diseases, facilitating their transmission. In many developing nations, including India, there is limited documentation on the extent of soil contamination, which poses challenges for implementing effective One Health strategies aimed at controlling these infections. Aim: To study the molecular prevalence and distribution pattern of pathogenic protozoa in soil from North India. Methods: In this prospective cross-sectional study, we collected a total of 200 soil samples from various locations in the Chandigarh region, including public parks, urban slum areas, villages on the outskirts, and sites near cattle sheds, hospitals, and public schools. DNA extraction was done in all the samples followed by conventional and nested PCR for the identification of Toxoplasma gondii, Entamoeba histolytica, Giardia duodenalis, and Cryptosporidium species from these samples. Key results: Out of 200, 34 soil samples (17%) from different sites in Chandigarh were found to have pathogenic protozoan contamination. Toxoplasma gondii was the most common protozoa (n = 18), followed by Entamoeba, Giardia, and Cryptosporidium species. Urban slums and public parks were most likely to be contaminated with oocysts/cysts of pathogenic protozoa. Conclusions: This study is the first of its kind in India, and the prevalence of oocyst/cyst of protozoa in our city was found significant. The findings underscore the critical role of soil contamination by pathogenic protozoa as a potential source for spreading zoonotic infections. © 2024 Elsevier B.V., All rights reserved.