In this study, antimicrobial activity of the essential oil (EO) and extracts of Himalayan-thyme (Thymus linearis Benth.) was evaluated against nine pathogenic microbial strains using disc diffusion and microdilution broth assays. The chemical composition of the EO was determined by gas chromatography-flame ionisation detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). The molecular mechanism of action of the EO was investigated against three pathogenic bacteria (Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis) and three fungal strains (Candida albicans, Candida glabrata, and Candida tropicalis) using the assays of protein leakage, and DNA and RNA leakage through the membrane. Major components of the EO were thymol (54.9%), γ-terpinene (16.6%) and p- cymene (5.2%). The EO showed broad-spectrum potent antibacterial and antifungal activity (MIC 240â990 µg/mL) against test strains. The EO damaged cell membranes and altered cell membrane permeability of test microorganisms. The EO showed a time- and dose-dependent bactericidal and fungicidal effect. It could act as a promising antimicrobial agent due to its short killing time.