The use of combined essential oils (EOs) is a new technique that can improve their preservative effects while minimizing their sensory impact in foods. The aim of this study was to determine the chemical profile of three essential oils (EOs) extracted from Lavandula stoechas L. (Ls), Thymus zygis L. (Tz), and Eucalyptus camaldulensis Dehnh (Ec) and to evaluate their synergistic antibacterial activity for optimal inhibition against Bacillus subtilis, Escherichia coli, and Staphylococcus aureus using an augmented Simplex centroid mixing scheme. The essential oils were extracted by hydrodistillation and analyzed via gas chromatography–mass spectrometry. Anti-bacterial potency was evaluated by disk diffusion. Chemical analysis revealed the main compounds in Lavandula stoechas (Ls) essential oil: camphor (36.15%), followed by fenchone (16.57%) and Z-8-hydroxy linalool (8.28%). The Thymus zygis (Tz) essential oil is dominated by δ-terpineol (27.64%), δ-3-carene (15.7%), and thymol (14.17%). In contrast, the Eucalyptus camaldulensis (Ec) essential oil contains mainly 1,8-cineole (43.61%), γ-terpinene (11.71%), and α-terpineol (10.58%). The optimal mixture is the binary association of 40% E. camaldulensis EO and 60% T. zygis EO, which provides an effective inhibition diameter (ID) of 13.37 mm to inhibit S. aureus. Furthermore, the formulation of 27% and 73% EOs of E. camaldulensis and T. zygis, respectively, corresponds to the mixture required to achieve the optimum inhibition diameter (ID = 11.55 mm) against E. coli. In addition, the mixture of 29% EO of E. camaldulensis and 71% EO of T. zygis is the optimum mixture to inhibit B. subtilis, with an inhibition diameter of 12.31 mm. These findings highlight the potency of antibacterial formulations of these essential oils and suggest that they might be used as substitutes for conventional drugs to prevent the development of bacteria responsible for serious infections and food spoilage.
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