Essential oils have many important biological properties, including antibacterial and antibiofilm activities. These unique properties make, essential oils good alternatives to synthetic chemical drugs, which have many side effects. In this study, we aimed to determine the chemical composition and biological activity of the essential oil obtained from Scutellaria brevibracteata subsp. brevibracteata. Specifically, its antibiofilm activity against Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 29213 biofilms using XTT assay. Cytotoxic and apoptotic properties of the essential oil were investigated in human lung cancer cells (A540 and H1299) using MTT assay, Annexin V‐FITC and propidium iodide staining and q‐PCR. Thirty‐two different compounds were identified from the essential oil, of which elemol (20.42%), γ‐eudesmol (20.12%) and β‐eudesmol (14.85%) were the main components. The essential oil was more effective against P. aeruginosa PAO1 biofilm (79%) than S. aureus ATCC 29213 biofilm (27%). The specific activity of the essential oil against P. aeruginosa biofilm may be related to its high terpene contents. In addition, the essential oil showed high cytotoxic activity towards A549 (IC50 9.09 µg/ml) and H1299 (IC50 55.04 µg/ml) cell lines, inducing apoptosis in these cancer cells. These results demonstrate the antibiofilm and anticancer activities of S. brevibracteata subsp. brevibracteata essential oil.