This study aimed to investigate the endophytes of Juniperus macrocarpa collected from ?esme in ?zmir, Turkey, using a culture-dependent approach and to evaluate their antimicrobial activity for the first time. Since endophytes interact with phytochemicals of the host plant, in addition to the standard culture media, a J. macrocarpa extract supplemented culture media was also used for isolation to enhance the cultivability of the endophytes. Six bacteria out of twelve and three fungi out of seven were isolated from the plant extract supplemented culture media. The genotypic identification of the bacterial and fungal isolates was determined based on 16S rDNA and Internal Transcribed Spacer (ITS) sequence analysis, respectively. The genus Juniperus, which has ethnopharmacological uses, is rich in phytochemicals with multiple bioactivities. Since Juniperus spp. is listed as a priority natural habitat, it is necessary to find alternative resources that could replace the bioactive compounds of these plants. Endophytes of Juniperus spp. might be good candidates as antimicrobial producers. From this point of view, the antimicrobial activity of the crude fermentation liquid of the J. macrocarpa endophytes, and also aqueous and methanolic extracts of J. macrocarpa, were evaluated using a disc diffusion assay against a panel of test microorganisms, including antibiotic resistant ones. One fungus and seven bacteria showed remarkable antimicrobial activity against at least one test microorganism. These results indicated that some endophytes of J. macrocarpa had antimicrobial properties like their host plant and could substitute these plants as a source of antimicrobials.