Background: Vitex agnus-castus is a deciduous shrub that is native to the Mediterranean region. It has traditionally been used in Iranian medicine. In the current study, Vitex agnus-castus Essential Oil (EO) leaves were analyzed for their chemical component as well as antioxidant and antifungal activity. Objectives: The aim of this study was to determine the biological properties (phytochemical component, and antioxidant and antifungal activity) of Vitex agnus-castus EO from an Iranian origin. Materials and Methods: Chemical composition of the EO was determined by using Gas Chromatography (GC) and Gas Chromatography/ Mass Spectrometry (GC-MS). Antioxidant activity of the Vitex agnus-castus EO was examined by the 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) assay, while the total phenolic content was also determined. Antifungal activity (against Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida dubliniensis, Aspergillus flavus, Aspergillus niger, Penicillium species and Alternaria species) was performed by a broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) protocols M27-A and M38-A for yeasts and filamentous species. Results: Thirty-two components were identified in Vitex agnus-castus EO. The main compound was alpha-Pinene (19.48%). The total phenolic content of EO was determined as 82.26 ± 5.94 mg Gallic Acid Equivalent (GAE)/g EO. The EO exhibited significant radical scavenging activity with IC50 value of 27.16 µg/mL. The obtained EO showed significant antifungal activity. Aspergillus niger was more susceptible than other fungi (MIC: 0.78 µL/mL). Conclusions: Potent antifungal activity, make this plant an effective replacement treatment for fungal infections or fungal strains that are resistance to synthetic antifungals.