The following study deals with the chemical composition, antimicrobial activity of essential oils of Vitex agnus-castus L. and their main constituents in vitro and in vivo. The main compounds in the oil of unripe fruits were sabinene (17.8%) and 1,8-cineole (17.5%), while in the oil of the ripe fruits dominant compounds were 1,8-cineole (16.3%) and sabinene (13.4%). The leaves oil contained an abundance of 1,8-cineole (22.0%), as well. All of the oils tested were rich sources of α-pinene (12.2%, 9.4% and 9.4%, respectively). Antimicrobial activity was tested using bacterial and fungal strains by the microdilution method. Using the same technique 1,8-cineole and α-pinene showed very high antimicrobial potency as well. As 1,8-cineole was the predominant constituent of the oils, we have chosen to test it further in an in vivo experiment. Randomly chosen apples were treated with 1,8-cineol solution and infected with Aspergillus niger in order to provoke Aspergillus rot in apples. Disease incidence was recorded.Highlights► Essential oils of Vitex agnus-castus were reach source of 1,8-cineole. ► Antimicrobial potency of essential oils, 1,8-cineole and α-pinene was high and comparable with control antimicrobial agents. ► 1,8-Cineole completely inhibited Aspergillus rot development in apple fruits.