The association between Toxoplasma gondii (T. gondii) infection and malignancy has attracted increased attention in recent years, but little is known of T. gondii infection among women diagnosed with a gynecological tumor (GT) in China. We conducted a case-control study involving 460 women diagnosed with a GT and 460 age-matched healthy controls (HCs) to estimate the infection process of T. gondii and understand the risk factors of T. gondii infection in patients with a GT. Levels of anti-T. gondii IgG and IgM were measured by enzyme-linked immunoassays every 12 months. After a median follow-up time of 4.3 years (range 4 to 5 years), 55/460 (11.96%) patients with a GT and 15/460 (3.26%) HCs were seroprevalence for T. gondii antibodies, respectively (P = 0.001). IgG antibodies against T. gondii were found in 54 GT patients (11.74%) and 15 HCs (3.26%), respectively (P = 0.001). The seroprevalence of T. gondii IgM antibodies was similar in patients with a GT and with HCs (2.83% vs 1.3%, P = 0.105). Multivariate stepwise logistic regression analysis revealed contact with cats (OR, 6.67; 95% CI [2.89–10.75]; P = 0.001), exposure to soil (OR, 2.16; 95% CI [1.14–4.10]; P = 0.019), being a farm-worker (OR, 4.17; 95% CI [1.20–11.49]; P = 0.006) and history of chemotherapy (OR, 3.16; 95% CI [1.56–6.45]; P = 0.001) to be independent risk factors for T. gondii infection. Women with an ovarian cancer or endometrial cancer had higher T. gondii seroprevalence than that of HCs. Moreover, T. gondii infection in patients with a GT mostly acquired within two years of diagnosis, but the infection in healthy controls had no obvious time characteristics. Here, we demonstrated that T. gondii infection is significantly higher in patients with a GT (especially in women with an ovarian tumor) compared to HCs. Thus, infection with this parasite should be avoided in patients with a GT, and the causal relationship between T. gondii and GTs should be studied in detail.