Background: Toxoplasmosis is caused by Toxoplasma gondii, a protozoan parasite, and is among the diseases generally referred to as neglected tropical disease (NTD). It can be a reason for impulsive abortion in pregnant women or hereditary chorioretinitis in neonates. Objectives: The present study examined seroprevalence and related risk issues of toxoplasmosis in pregnant women (PTW) and non-pregnant females (NPF) of two health facilities in Port Harcourt. Methods: Venous blood was collected from 380 women and analyzed by immunodiagnostic techniques using ELISA (Enzyme-Linked Immunosorbent assay) IgG and IgM tests and real-time polymerase chain reaction (PCR). Data were collected using a well-structured questionnaire on the socio-demographic risk issues linked with toxoplasmosis. Results: Among the study population, PTW and NPF had seroprevalence of 33.0% and 19.4%, respectively (P < 0.05). PTW recorded a higher overall seroprevalence than NPF with 16.1%, 1.3%, and 8.4%, 0.8% for Toxoplasma gondii ELISA IgG, and IgM tests, respectively. IgM-positive cases were all authenticated by the real-time PCR technique and were all negative. Age group 35-39 years had the highest IgG seroprevalence of 24.3% for PTW, while the age group > 40 years had the highest IgG seroprevalence of 18.2% for NPF. Traders recorded the uppermost seroprevalence of 20.9% and 2.6% for IgG and IgM among PTW, respectively, while teachers and traders recorded the maximum seroprevalence of 15.6% and 1.7% for IgG and IgM among NPF, respectively. Statistical analysis showed that there was no association between the age group, occupation, trimester, and toxoplasmosis. Conclusions: The toxoplasmosis was significantly high in pregnant women. It is recommended that Toxoplasma gondii tests should be included as a routine test during antenatal.