Background: In recent years, there has been an increase in the prevalence of Enterococcus faecalis strains with acquired antimicrobial resistance in the oral environment, a phenomenon associated with horizontal gene transfer. The oral cavity can act as a reservoir and a critical point for the persistence and transmission of multidrug-resistant microorganisms, posing a challenge to public health. Aim: To identify genes associated with acquired antimicrobial resistance and determine their prevalence against selected antibiotics as epidemiological surveillance markers in Enterococcus faecalis strains isolated from patients who attended endodontic consultations. Methods: Microbiological identification and antimicrobial susceptibility profiling were evaluated using the Dade/MicroScan Pos ID PC34 system (West Sacramento, CA, USA), in accordance with CLSI standards. PCR using specific primers allowed the detection of acquired resistance genes for epidemiologically relevant antibiotic markers in the oral flora, including erythromycin (ERY), high-level gentamicin resistance (HLGR), high-level streptomycin resistance (HLSR), tetracycline (TET), and vancomycin (VAN). Results: 35.3% of Enterococcus faecalis isolates showed acquired resistance, with particular emphasis on resistance to GEN (11.8%), HLSR (14.7%), TET (5.9%) and ERY (2.9%). VAN resistance was absent. The genes mainly associated with this resistance were ermB, tetM and aac(6')-Ie-aph(2")-Ia. Conclusion: The presence of acquired resistance genes in E. faecalis from endodontic infections was evident, signalling the oral cavity as an antimicrobial reservoir. These findings underscore the need for microbiological surveillance in dentistry.