The objective of this study was to assess the biological activity of essential oils (EOs) of four species obtained via two different distillation methods and their potential as biopesticides. The studied factors were juniper species ( L., L., C. Koch., and Burgsd), plant sex (male (M) and female (F)), and distillation method (hydrodistillation via a standard Clevenger apparatus (ClevA) and semi-commercial (SCom) steam distillation). The hypothesis was that the EO will have differential antioxidant, antimicrobial, and insecticidal activities as a function of plant species, plant sex, and distillation method. The two distillation methods resulted in similar EO composition within a given species. However, there were differences in the EO content (yield) due to the sex of the plant, and also differences in the proportions of some EO components. The concentration of α-pinene, β-caryophyllene, δ-cadinene and δ-cadinol was dissimilar between the EO of M and F plants within all four species. Additionally, M and F plants of , and had significantly different concentrations of sabinene within the respective species. The EOs obtained via ClevA extraction showed higher antioxidant capacity within a species compared with those from SCom extraction. All of the tested EOs had significant repellent and insecticidal activity against the two aphid species (bird cherry-oat aphid) and (English grain aphid) at concentrations of the EO in the solution of 1%, 2.5%, and 5%. The tested EOs demonstrated moderate activity against selected pathogens spp., , spp., and . The results demonstrate that the standard ClevA would provide comparable EO content and composition in comparison with SCom steam distillation; however, even slight differences in the EO composition may translate into differential bioactivity.