Salmonellosis is an infectious disease affecting both animals and humans. Antimicrobial resistant (AMR) and biofilm-producingSalmonellaspp., frequently detected in reptiles (who can then act as asymptomatic carriers for warm-blooded animals), have developed resistance to biocides; this represents a warning for the emergence of biocide/antimicrobial cross-resistance. The aim of this study was to evaluate the efficacy ofThymus vulgarisL. essential oil (TEO) in inhibiting bacterial growth and biofilm production ofSalmonellaspp., which had been isolated from wild reptiles housed in a Zoo in Italy. The resistance profile against different classes of antibiotics showed that all the isolates were susceptible to the tested antibiotics, despite the presence of several AMR genes. All the isolates were also tested with aqueous solutions of TEO at different dilutions (5% to 0.039%). Interestingly, TEO proved effective both in inhibiting bacterial growth at low dilutions, with MIC and MBC values ranging between 0.078% and 0.312%, and in inhibiting biofilm production, with values ranging from 0.039% to 0.156%. TEO demonstrated effective bioactivity against the biofilm producerSalmonellaspp., proving to be a valid disinfectant for the prevention of salmonellosis from reptiles, a possible source of infection for humans exposed to the reptiles’ environment.