The epidemiological importance of the different routes of Toxoplasma gondii transmission is not known and depends largely on population behaviour and knowledge. This study was conducted to assess toxoplasmosis seropositivity and the related knowledge and preventive practices that are necessary for the prevention of the disease among pregnant women. All pregnant women attending antenatal clinic were tested for T. gondii immunoglobulins followed by a survey questionnaire that tested their knowledge and preventive practice. Statistical comparisons were made between the seropositive and negative ones. We determined a low to moderate seroprevalence of toxoplasmosis among pregnant women in Dhahran, Saudi Arabia as compared to many other parts of the world. The overall positivity rates of IgG and IgM against T. gondii among 400 pregnant women were 28.5 and 3%, respectively. 75.5% of the participants had never heard about toxoplasmosis and the associated risk factors. Lack of knowledge was associated with the higher risk of infection (OR = 4.04, p < 0.001). Keeping pet cats was not common and poorly associated with infections (OR = 1.15, p ≥ 0.64). Consumption of undercooked meat was reported frequently and only slight risk was associated with sheep/goat meat (OR = 1.39, p = 0.15). Eating outside the home at restaurants was reported for the first time to be related to a higher risk of infection (OR = 2.69, p < 0.001). Several possible risk factors were suggested through odds ratios calculation and overall knowledge of toxoplasmosis by pregnant women was poor. It is therefore vital to provide a formal education about toxoplasmosis risk factors to women of childbearing age.