The aim of the present study was a chemical, microbiological and statistics analysis of Hyssopus officinalis L. subsp. aristatus (Godr.) Nyman essential oil during different stages of development from three natural populations of plant collected in southeastern Serbia. In addition, using statistical tools, we also tried to explain the effect of dominant componentsˈ content on antimicrobial activity. Oxygenated monoterpenes were the most abundant compound class in the H. officinalis oil during examined phenological stages (27.32‐92.25%). The four most abundant essential oil components are eucalyptol, cis‐pinocamphone, β‐pinene and β‐ocimene. H. officinalis essential oils demonstrated minimum inhibitory concentrations and minimum microbicidal concentrations values from 2.4 to 160 mg/mL. Maximum antimicrobial activity, in total, was recorded in November, after the flowering stage. The percentage of dominant compounds of essential oils, eucalyptol, and cis‐pinocamphone, affects essential oils' antibacterial activity. The essential oil with most closely matched research criteria was extracted from a plant in Kravlje village.