Introduction: This preliminary cross-sectional study aimed to investigate the prevalence of toxoplasmosis among blood donors in Kelantan, Malaysia. Methods: A total of 56 blood donors were screened by an enzyme-linked immunosorbent assay (ELISA) for anti-T. gondii Immunoglobulin G (IgG) and Immunoglobulin M (IgM) antibodies. Positive T. gondii IgG and IgM were further tested for IgG avidity ELISA. All extracted deoxyribonucleic acids (DNAs) from whole blood samples were analyzed for the presence of the Toxoplasma B1 gene and the ITS1 region by polymerase chain reaction (PCR). The socio-demographic data of donors was assessed using a data collection form. Results: Out of 56 blood donors, 24 (42.86%) donors were IgG+/IgM-, and 2 (3.57%) donors were IgG+/IgM+ with one of them having a high avidity index indicating as past infection for more than 20 weeks and the other with a low avidity index indicating as recent infection within 20 weeks. None of the samples tested positive for the presence of the Toxoplasma B1 gene and the ITS1 region. A univariate analysis showed that only employment status was significantly associated with Toxoplasma seropositivity. Conclusion: The seroprevalence of toxoplasmosis among blood donors in Kelantan, Malaysia, was 46.43%. Nevertheless, direct detection by PCR showed that this parasite was absent in the blood. These results highlight that the blood donors in this study had previously been exposed to T. gondii infection. The parasite may still remain in certain tissues but does not freely circulate in the blood.