Introduction: Cats serve as definitive hosts for several zoonotic parasites, most notably Toxoplasma gondii, which can contaminate the environment with infective oocysts and pose significant risks to public health. Shelter populations, often composed of stray, rescued, and previously owned cats, represent an important reservoir of infection due to their diverse backgrounds and variable health conditions. Material and Methods: In this study, a total of 156 fecal samples were collected from shelter cats in Bogor, Indonesia, comprising 102 adults and 54 kittens. Samples were examined using direct microscopy, modified Ziehl–Neelsen (MZN) staining, and nested PCR targeting the 5′ SAG2 and GRA7 genes. Statistical analysis was performed using the Chi Square test to evaluate associations between age and parasite prevalence. Results: Overall, intestinal parasites were detected in 25.64% of samples. Hookworms were the most frequently identified (10.25%), followed by T. gondii oocysts (9.61%), Toxocara spp. (5.12%), and Cystoisospora spp. (0.64%). The prevalence of T. gondii oocysts was significantly higher in kittens compared to adults (p < 0.05), underscoring the role of younger cats in environmental contamination. Of the 15 microscopy positive samples, molecular confirmation identified four positives for the 5′ SAG2 gene and two for the GRA7 gene, thereby validating the presence of T. gondii and reducing the likelihood of misidentification with morphologically similar protozoa. Conclusion: These findings highlight shelter cats, particularly kittens, as an important source of zoonotic parasites. The integration of parasitological and molecular methods enhances detection accuracy and provides a more reliable assessment of infection dynamics. Strengthened surveillance and targeted control strategies are essential to mitigate environmental contamination, reduce zoonotic transmission, and protect both animal and human health. |