Antimicrobial preservatives are widely added to health products to prevent microbial contamination. However, because of the risks attributed to several synthetic agents, manufacturers are searching for new natural agents. Therefore, this work investigated the different physicochemical characteristics of one of those natural ingredients, the essential oil (EO) of Moroccan thyme (Thymus satureioides C.) and its antimicrobial potential against five microbial strains. The physicochemical parameters (density, refractive index, optical rotation, miscibility, acid value, ester value, and flash point) were measured and the chemical composition was determined by GC/MS. The antimicrobial activity was assessed using disc diffusion method and a macrodilution broth method. The EO yield was 1.01% compared to the dry matter. Fifty-two compounds were identified. The major compounds were thymol (28.66%), borneol (21.16%) and α- terpineol (12.33%). The disc diffusion method revealed that all the strains tested showed sensitivity to the EO at concentration of 1636 μg per disc. For the minimum inhibitory concentration (MIC) and the minimum microbicidal concentrations (MMC), similar results were obtained for Staphylococcus aureus and Escherichia coli (640 μg/mL), for Pseudomonas aeruginosa (960 μg/mL) and for Candida albicans (800 μg/mL) baring Aspergillus brasiliensis, which had 480 μg/mL and 640 μg/mL for the MIC and MMC, respectively.