Fleas of the genus Ctenocephalides that infest cats and dogs are known to harbor various pathogenic bacteria with potential zoonotic importance. This study aimed to isolate, characterize, and identify bacterial species from the gastrointestinal tracts of Ctenocephalides felis and Ctenocephalides canis using biochemical profiling, antibiotic susceptibility testing, and 16S rRNA gene sequencing. A total of ten bacterial isolates were obtained, showing different biochemical and antibiotic resistance profiles. Biochemical identification revealed the presence of Staphylococcus, Pseudomonas, Francisella, and Sphingomonas species, while molecular identification indicated variations at both the genus and species levels. Notably, isolates from C. canis exhibited higher resistance compared to those from C. felis, with Benzylpenicillin being the most resistant antibiotic. These findings highlight the species divergence and antibiotic resistance patterns of flea-associated bacteria, emphasizing their potential role in zoonotic transmission and the need for integrated monitoring of flea-borne pathogens.