The prevalence of Toxoplasma gondii infection in ruminants (sheep, goats, and cattle) in Southern Tunisia is largely unknown. Antibodies and DNA to T. gondii were determined in serum and apex of the heart samples of 261 animals (204 sheep, 32 goats and 25 cattle) using the modified agglutination technique test (MAT) and the PCR respectively. Antibodies to T. gondii (MAT, 1:20) were found in 40.2% (95% CI: 33.4%, 47.2%), 34.5% (95% CI: 19.1%, 53.2%) and 12% (95% CI: 03.15%, 32.13%) in sheep, goats and cattle respectively. Seroprevalence significantly (p<0.05) varied with species, gender, age, and animals’ breed. Older animals (>3 years) and females were significantly (p<0.05) more infected than younger and male animals respectively. The seroprevalence was highest in sheep and specifically in the Barbarine breed. The presence of Toxoplasma DNA was detected in 11 of 96 samples (11.5%). The prevalence observed in the present study indicates a widespread exposure to T. gondii in South Tunisia. Results indicate, for the first time in Tunisia, that infected sheep and goats represent a potential source of T. gondii infection for humans in particular in the South of the country (Gafsa area).