Although African lions (Panthera leo) are listed as vulnerable species, it is a famous practice in certain nations to display them in the circus. To date, the gastrointestinal bacterial flora of lions is poorly understood. This study aimed to clarify the zoonotic potential isolation of the bacteria posed by lions of the Egyptian national circus along with PCR screening for their virulence and antibiotic resistance genes. Nine captive African lions were examined bacteriologically. Clostridia spp. was the most prevalent bacteria (55.5%) in lions, then Staphylococcus spp. (33.3%), followed by E. coli and Edwardsiella spp. (22.2% for each) and Serratia spp. (11.1%). The isolated bacteria showed multiple drug resistance patterns of multiple antibiotics (ampicillin, cefepime, cefoxitin, piperacillin, amoxicillin, chloramphenicol, ceftazidime and tetracycline). Two isolates of C. perfringens were positive for Alpha toxin and bla resistant genes using PCR. Also, all S. aureus isolates were confirmed by the presence of 16s rRNA gene and exhibited the virulence (spa, and coa) and resistance (blaZ) genes. Additionally, the two isolated E. coli strains were positive for virulence (phoA, eaeA) and resistance (Aada1) gene but, Aada2 was completely absent. In conclusion, this study is actually unprecedented and provides a start for the upcoming research to investigate the current health status of captive wild animals exhibited at the circuses.