In this study, 19 endophytic fungal isolates associated with Artemisia judaicaL. were isolatedfrom Al-Shihiyah, Haʾil, Saudi Arabia. Those isolates represented five genera, including: Alternaria sp., (predominantly), Aspergillus sp., Chaetomium sp., Cladosporium sp., and Fusarium sp. All isolates (6 species) were grown on a solid rice medium and their ethyl acetate crude extracts, beside A. judaica L. extract, were tested for their antimicrobial activity. One endophytic fungal isolate demonstrated the highest activity and was chosen for further investigation. Based on its phenotypic, ITS ribosomal gene sequences, and phylogenetic characterization, this isolate was identified as Alternaria tenuissima AUMC14342 with accession number (MT468650). Ethyl acetate (EtOAc) extract from the isolated strain, AUMC14342, was fractioned using VLC. The active fraction of the EtOAc exhibited good antimicrobial effect against P. aeruginosa, S.aureus, F. solani, and A. niger at conc. 30 µ/mL using disc diffusion assay. A moderate cytotoxic effect was reported against a Hepatocellular carcinoma cell line (HepG2), with the lowest inhibitory concentration (IC50) of 0.52 µM. The active fraction was characterized by the presence of different chemically terpene, ester, volatile, and other organic compounds, determined by GC–MS. Conclusively, the endophytic fungus, A. tenuissima, derived from A .judaicaL.,is an ideal candidate for the potential industrial production of antimicrobial agents with a variety of applications.