Considering the increasing popularity of reptiles as pets and their possible role as reservoirs of pathogenic microorganisms, the aim of this study was to isolateEscherichia coli, Salmonellaspp., Clostridium perfringens, andC. difficilestrains from reptiles in Brazil and to characterize the isolated strains. The characterization was based on phylogenetic typing ofE. coli, identification of virulence genes ofE. coli, C. perfringens,andC. difficile, serotyping ofSalmonellaspp., ribotyping and MLST ofC. difficileand antimicrobial susceptibility test of pathogenic strains. Cloacal swabs were collected from 76 reptiles, of which 15 were lizards, 16 chelonians, and 45 snakes, either living in captivity, in the wild, or as companion animals.E. coliwas isolated from 52 (68.4%) reptiles, of which 46 (88.4%) were characterized as phylogroup B1. The virulence factor CNF1 ofE. coliwas found in seven (9.2%) sampled animals, whereas the gene of EAST1 was found in isolates from two (2.6%) reptiles. Three isolates positive for CNF1 were resistant to cephalothin, one of which was also resistant to ciprofloxacin, trimethoprim/sulfamethoxazole, and chloramphenicol, being then classified as multidrug resistant strain (MDR).Salmonella entericawas identified in 26 (34.2%) reptiles, of which 13 belonged to the subspeciesenterica.Serotypes such asS.Mbandaka,S.Panama,S. Infantis,S.Heidelberg, andS.Anatum were identified. One isolate ofS. entericasubsp.houtenaewas resistant to cephalothin and ciprofloxacin.C. perfringenstype A was isolated from six (7.8%) animals.C. difficilewas isolated from three (3.9%) reptiles. Two of these isolates were toxigenic and classified into ribotypes/MLST 081/ST9 and 106/ST42, which have been previously reported to infect humans. In conclusion, reptiles in Brazil can harbor toxigenicC. difficileand potentially pathogenicE. coliandSalmonella entericasubsp.enterica, thus representing a risk to human and animal health.