This study was performed to investigate the constituents, in vitro antifungal activity and phytotoxicity potential of the essential oil from Juniperus polycarpos var. turcomanica leaves. The essential oil was analyzed by GC–FID, and GC/MS, which predominantly contains a-pinene (51.21%), germacrene–B (4.80%), and D-cadinene (2.56%). The antifungal activity of the essential oil against some phytopathogenic fungi, including Alternaria alternata, Colletotrichum trichellum, Curvularia fallax, Cytospora sacchari, Fusarium oxysporum, and Macrophomina phaseolina was performed through disk diffusion and agar dilution assays. The essential oil of J. polycarpos var. turcomanica had high antifungal activity against tested phytopathogenic fungi. The most susceptible fungi to the essential oil were C. trichellum in agar dilution and M. phaseolina and C. fallax in disk diffusion methods, whereas, the most resistant fungus to the essential oil was obtained from A. alternata in both assays. Screening methods had an influence on antifungal activity of the essential oil as most of the tested fungi in this study were shown to be more resistant in disc diffusion methods. According to the phytotoxic assay results, the essential oil from J. polycarpos var. turcomanica had high phytotoxicity against three species of weeds, including P. oleracea L., A. retroflexus L., and D. stramonium L. The results of this research suggest that the herbicidal and antifungal activities of the essential oil from J. polycarpos var. turcomanica can be attributed to its major group of constituents, monoterpenes hydrocarbons.