Antimicrobial-resistance (AMR) is a ‘natural phenomenon’ based on a selection of microorganisms able to survive in an unfavourable environment, owing to genetic mutations or the acquisition of “pre-established” resistance genes. The overuse and misuse of antibiotics can favour the emergence and spread of AMR, with negative impacts on the management of bacterial infections and economic implications for healthcare systems. Research and development of natural antibacterial molecules, which exhibit multiple bio-functionalities and are less likely to induce resistance in bacteria, could represent a priority in the coming years to improve the antibacterial activity of existing molecules and counteract AMR. The present study identified the most effective concentration and contact time of Thymus vulgaris L. essential oil (TEO) to achieve in vitro bactericidal activity against twenty-one bacterial strains isolated from different specimens. In total, 10 μl of a solution containing the TEO and the bacterial strains isolated was sown in Petri-dishes for successive assessments of antibacterial efficacy, in terms of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), after 24 and 48 hours, respectively. The identified strains (Mammaliicoccus lentus, 2Escherichia coli, Salmonella enterica subsp.enterica sierovar derby, 2Staphylococccus, S.xylosus, S.chromogenes, S.epidermidis, S.enterica subsp.diarizonae, S.enterica subsp.salamae, S.enterica subsp.houtenae, E.coli(b), S.aureus(b), Citrobacter freundii, Enterococcus feciorum, Proteus mirabilis, Acinetobacter cioffi, Pseudomonas putrefaciens and Klebsiella pneumoniae), each with different resistance profiles, and two ATCC strains (S.aureus and Streptococcus mutans) were tested after 6, 12, 24, and 48h of contact with TEO at different concentrations, from 5% to 2.5% to 1.25%(v/v), corresponding to 450, 225, and 112.5g/mL, respectively. We observed a complete inhibition of all bacterial strains after 12h of incubation at all TEO concentrations, demonstrating the efficacy of TEO against several Gram-positive and Gram-negative bacteria with different AMR. Further studies are needed to define the exact molecular mechanisms of TEO and its possible uses.