Background: Patients undergoing hemodialysis are considered to be immunocompromised. Hence, these individuals are more vulnerable to opportunistic pathogens such as Toxoplasma gondii (T. gondii). This study aimed to assess seroprevalence of T. gondii infection among patient undergoing hemodialysis in Qalyubia Governorate, Egypt. Methods: This observational, comparative cross-sectional study included 150 participants: 100 patients undergoing hemodialysis and 50 healthy control subjects. The study was conducted in the hemodialysis units located in Qalyubia Governorate. Results: Logistic regression analysis revealed that patients undergoing hemodialysis had significantly higher odds of IgG seropositivity compared to the control group, with an adjusted odds ratio (aOR) of 3.09 (95% CI: 1.47–6.48, P