The increasing resistance of pathogenic bacteria to conventional antibiotics is a major public health problem. Thus the need for other alternatives in the search for new biologically active molecules is increasing. The study of the antibacterial activity of essential oils is one of the promising ways because of their antibiotic proven effectiveness on the one hand and the diversity of their biochemical composition conferring a broad spectrum of biological activities with mechanisms varied activities on the other. This work has focused on the study of the essential oil of biological activity of sawdust burl Thuya (Tetraclinis articulata (Vahl) Masters) against tested reference and clinical strains of Staphylococcus aureus and Escherichia coli. This study aimed to the recovery of waste from the processing of Thuya burl wood in the region of Essaouira by extracting essential oil and evaluation of its biological activity against multi-resistant strains of bacteria. The essential oil of sawdust of Thuya burl wood was obtained by steam distillation with a yield of about 1.5%. The antibacterial activity was evaluated using the aromatogram method showed a high activity of the essential oil against the tested strains. This activity was 2 to 6 times greater than that obtained with the reference antibiotic. Furthermore, this essential oil was more effective on Staphylococcus aureus (Gram+) and Escherichia coli (Gram-) with respectively bacteriostatic and bactericidal activities.