This study estimated the seroprevalence and risk factors for acquiring Toxoplasma gondii infection by undergraduate female university students in Jordan. A cross-sectional study from September 2013 to July 2014 analysed 202 blood samples for IgG and IgM antibodies by enzyme-linked immunosorbent assay and a semi-constructed questionnaire was completed by participants to gather information about Toxoplasma infection risk factors. T. gondii IgG antibodies were detected in 66·5% of the females. Only one sample was positive for both IgG and IgM. Using χ2 test, six factors showed significant association with T. gondii infection (P ⩽ 0·01). The multivariate logistic regression model showed that female students living in houses, wet areas, with income >US$750/month and using spring (untreated) water were 47·42, 10·20, 5·00, 3·25 more times at risk to be seropositive for T. gondii, respectively, compared to female students living in apartments, dry areas, with income ≤US$750/month and using treated water, respectively. This study concluded that T. gondii infection in female university students in Jordan is high and most women become infected before marriage; however, congenital toxoplasmosis is still likely to occur in Jordan. Thus, dissemination of protective measures and knowledge by healthcare professionals is essential especially for pregnant women.