Clostridium piliforme is an anaerobic, spore-forming, obligate intracellular bacterial pathogen that causes Tyzzer’s disease in laboratory, wild, and domestic animals. C. piliforme has significant economic implications for laboratory and commercial animal facilities due to its impact on research integrity, increased veterinary and management costs, and the need for enhanced biosecurity measures. In the present study, 100 rabbit faecal samples were collected, and nested PCR was performed using C. piliforme specific 16S rRNA primers. C. piliforme was detected in all five rabbit farms, with an overall prevalence of 40%. A statistically significant difference in prevalence was observed among farms. Male faecal samples accounted for only 25% of the total, and although more females (45.33%) were found to be infected, this difference was not statistically significant. Rabbits older than 5 months (42.05%) were more frequently infected than those younger than 5 months (25%), though this was not statistically significant. Breed, weight, rearing system, and feeding type did not influence prevalence. Because 16S rRNA primers may yield nonspecific amplicons, their use in detecting C. piliforme in samples, particularly faeces, should be interpreted in conjunction with clinical signs or gross lesions. Identification of C. piliforme specific gene target primers is urgently needed for effective screening of susceptible populations.