Chlamydia and Chlamydia-like bacteria are well known to infect several organisms and may cause a wide range of diseases, particularly in ruminants. To gain insight into the prevalence and diversity of these intracellular bacteria, we applied a pan-Chlamydiales real-time PCR to 1,134 veterinary samples taken from 130 Tunisian ruminant herds. The true adjusted animal population-level prevalence was 12.9% in cattle, against 8.7% in sheep. In addition, the true adjusted herd-level prevalence of Chlamydiae was 80% in cattle and 25.5% in sheep. Chlamydiales from three family-level lineages were detected indicating a high biodiversity of Chlamydiales in ruminant herds. Our results showed that Parachlamydia acanthamoebae could be responsible for bovine and ovine chlamydiosis in central-eastern Tunisia. Multivariable logistic regression analysis at the animal population level indicated that strata and digestive disorders variables were the important risk factors of bovine and ovine chlamydiosis. However, origin and age variables were found to be associated with bovine and ovine chlamydiosis, respectively. At the herd level, risk factors for Chlamydia positivity were as follows: abortion and herd size for cattle against breeding system, cleaning frequency, quarantine, use of disinfectant and floor type for sheep. Paying attention to these risk factors will help improvement of control programs against this harmful zoonotic disease.