A cross-sectional study was conducted in 2024 to study the seroprevalence of Toxoplasma gondii (T. gondii) and male testosterone levels in Palestinian butchers in the West Bank. Sera were collected from 156 butchers and tested for anti-T. gondii-IgG and IgM using commercial immune assay and testosterone tests. Univariate and bivariate analyses were used to calculate crude and adjusted odds ratios. The seroprevalences were 62.8% for anti-T-gondii-IgG, 7.1% for anti-T-gondii-IgM, and 7.1% for both IgG and IgM. T. gondii-IgG seropositive individuals had a median testosterone level of 351 ng/dL, significantly lower than the 428.5 ng/dL observed in T. gondii-IgG seronegative participants (p = 0.017). T. gondii-IgG seropositivity was associated with raw meat consumption, exposure to soil, age, and years of practice with adjusted odd ratios of 3.85, 3.32, 1.10, and 1.27, respectively. However, low testosterone level was not significantly associated with T. gondii seropositivity (p = 0.07). This finding suggests that additional research is needed to understand the relationship between Toxoplasma infection and testosterone levels.