Toxoplasma gondii (T. gondii) affects around 30% of humans worldwide. Recently, it has emerged as a significant opportunistic pathogen to immunocompromised patients. Data available is still lacking about toxoplasmosis in cancer patients in Egypt. This study aimed to reveal the current trend of T. gondii in cancer patients in Sohag, Egypt. Sera from 50 cancer patients and 50 healthy controls were screened for Toxoplasma IgG and IgM. Further, buffy coats from both groups were used for detection of T. gondii B1 and RE genes via conventional and nested PCR, respectively. The overall seroprevalence of T. gondii IgG was high (58%). IgG and IgM were detected in 30% and 9% cancer patients, respectively. Patients with solid cancers exhibited a greater IgG seropositivity compared to those with hematologic tumors (77.27% and 46.43%, respectively) (P = 0.03). Concerning the molecular results, only 4 (9%) were positive regarding both PCR assays. In conclusion, T. gondii is highly prevalent in cancer patients in Sohag, Egypt. PCR is strongly recommended to complement serology to diagnose acute or reactivated toxoplasmosis in cancer patients. B1 PCR was found to be equivalent to RE PCR. Nevertheless, thorough large-scale research must be implemented.