The molecular and serological prevalence of Toxoplasma gondii infection was investigated among rodents living in desertic areas in the Tataouine district, in the south of Tunisia. A total number of 43 rodents were captured from four sites classified as arid and Saharan climatic zones. Sera, hearts, spleens and brains were collected from each rodent. Sera were tested for the presence of anti-T. gondii IgG by the ELISA technique. PCR was used to detect T. gondii DNA from different tissues. Two rodent species were identified as Ctenodactylus gundi (Rodentia, Ctenodactylidae) (N = 28; 65%) and Psammomys obesus (Rodentia, Muridae) (N = 15; 35%). The overall molecular prevalence of T. gondii was 39% (17/43). Infection prevalences were higher in C. gundi (53.6%; 15/28) compared to P. obesus (13.3%; 2/15). In both species, the brain was the most infected organ (p = 0.02). No significant difference was recorded for the two rodent species according to gender and sampling sites (p > 0.05). The overall seroprevalence was up to 34.9% (15/43). It was higher in C. gundi (43%; 12/28) compared to P. obesus (20; 3/15) (p = 0.02). These results highlight a high infection level of T. gondii in desertic rodents. More investigations are required to understand the role of other desertic mammals and to identify the genotype circulating in the Tunisian Sahara.