GC–MS Characterization and In Vitro Antibacterial Activity of a Standardized Multi–Essential Oil Formulation Containing Eucalyptus globulus, Thymus vulgaris, Zataria multiflora, and Mentha piperita Against Food Spoilage Bacteria

Background: Food spoilage bacteria significantly reduce shelf life and product quality, leading to economic losses in the food industry. Plant-derived essential oils are increasingly considered safe and sustainable alternatives to chemical preservatives due to their broad antimicrobial activity. Objective: This study aimed to evaluate the in vitro antibacterial activity of a standardized plant-based spray formulation against food spoilage bacteria and to characterize its chemical composition using GC–MS analysis. Materials and Methods: The standardized spray, obtained from Dineh Pharmaceutical Company (Iran), contained Eucalyptus globulus, essential oils of various Eucalyptus species, Thymus vulgaris, Zataria multiflora, Mentha piperita, menthol, ethanol (70%), and purified water. Antibacterial activity was assessed using agar well diffusion and macro-dilution methods against standard strains of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi, as well as clinical isolates of Klebsiella pneumoniae and Enterobacter aerogenes. All tests were performed in triplicate. Results: The formulation exhibited strong antibacterial activity, with the lowest minimum inhibitory concentration (MIC) observed against Staphylococcus aureus (7.81 µg/mL). Other tested microorganisms also showed high sensitivity. Conclusion: The standardized plant-based spray demonstrated broad-spectrum antibacterial activity, highlighting its potential application as a natural antimicrobial agent for food safety–related uses, including food packaging and surface sanitation.