The oral microbiota has a diversity of microorganisms that together maintain the homeostasis of the oral cavity. Disruptions in the balance of these microbial populations can lead to the development of oral and dental diseases. To characterize the normal oral microbiota of captive ophidians, samples were obtained from the oral cavity of eight Burmese pythons (Python bivittatus) and 11 royal pythons (Python regius), using oral swab, during routine evaluations. In the Laboratory-Antimicrobials, Biocides and Biofilms Unit at University of Trás-os-Montes e Alto Douro, the identification and antimicrobial susceptibility profile was determined using the Vitek^® 2 Compact automated device (bioMérieux, Marcy-l’Étoile, France). Of the 106 bacterial isolates obtained, 69% were Gram-negative species and 31% were Gram-positive bacteria. The genus Pseudomonas was the most frequently isolated. Chryseobacterium indologenes, Escherichia coli and Pseudomonas aeruginosa were the most isolated species. Antimicrobial susceptibility testing revealed that the phenotypic resistance was highest for nitrofurans (47.2%), beta-lactams (45.8%) and sulfonamides (30.6%). Twenty-one multidrug-resistant isolates (58.3%) were identified with Acinetobacter baumannii, Serratia plymuthica, Chryseobacterium indologenes, Providencia rettgeri and Pseudomonas aeruginosa showing the highest resistance frequencies.