•H. officinalis subsp. pilifer essential oil and deodorised extracts were studied.•1.8-Cineole, β-pinene and isopinocamphone were main compounds in the oil.•Syringic, chlorogenic, feruloylquinic, rosmarinic acids were characterized.•Essential oil and extract revealed lower antifungal activity than bifonazole.•DAE exhibited the best antioxidant activity and higher total phenolic content. The aim of the study was to investigate the chemical composition of Hyssopus officinalis L. subsp. pilifer (Pant.) Murb. essential oil (EO) and deodorized extracts (DE) and to evaluate their potential antifungal and antioxidant activities. EO was analyzed by GC–FID and GC–MS. Among the 30 compounds identified in the oil, the main were 1.8-cineole (36.43%), β-pinene (19.55%), isopinocamphone (15.32%) and trans-pinocamphone (6.39%). Deodorized aqueous extract (DAE) was analyzed by LC-DAD/ESI-TOF MS. The most significant phenolic acids detected in DAE were syringic, caffeoylqunic acids, feruloylquinic and rosmarinic acid. Aspergillus niger, A. ochraceus, A. versicolor, A. fumigatus, Cladosporium cladosporioides, C. fulvum, Penicillium funiculosum, P. ochrochloron, Trichoderma viride and Candida albicans yeast were used for antifungal assay. Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of the tested EO and DEs were recorded by the microdilution method. The antioxidant potential of the extracts was evaluated by means of the 2,2-diphenyl-1-picrylhydrazil (DPPH) radical scavenging method. The deodorized aqueous extract (DAE) possessed the highest scavenging activity (EC50=0.54mg/ml) followed by the deodorized methanol extract (DME) (EC50=0.82mg/ml) and deodorized ethyl acetate extract (DEE) (EC50=2.97mg/ml). EO possessed the lowest activities compared to other extracts and control substances. Total phenolic content of DEs was determined by Folin–Ciocalteau (FC) assay and gallic acid was used as the standard. DAE of H. officinalis subsp. pilifer exhibited the highest phenolic content 96.47 GAE.