The quest for new bioactive compounds from natural origins is continuously growing. In this study, Nine endophytic fungi were isolated and identified morphologically from healthy parts of the Azadirachta indica (neem) medicinal plant, and their crude extracts were evaluated for antimicrobial effectiveness against 12 pathogenic microorganisms, including bacteria, unicellular yeasts, and multicellular fungi. The cell-free filtrates were recovered using chloroform, diethyl ether, and ethyl acetate solvents. Ethyl acetate extraction of endophytic fungus E3 exhibited the highest antimicrobial activities using the response surface approach. The isolate was molecularly identified as Aspergillus terreus and deposited in the gene bank under accession number ON532722. In addition, the GC-MS analysis of normal and Gamma Irradiated A. terreus ethyl acetate crude extracts indicated the presence of 32 main bioactive components with diverse medicinal properties that demonstrate antioxidant scavenging activities of the DPPH free radical with an IC50 of 85.5 ± 3.62 µg/ml and 37.5 ±1.48 µg/ml for the normal and gamma-irradiated isolate respectively. Moreover, the bioactive substances demonstrated promising anticancer efficacy against human hepatocellular carcinoma (HepG-2) and lung carcinoma (A-549) with IC50 values of 20.74± 1.08 µg/ml, 26.89 ± 1.63 µg/ml, 48.91 ± 3.85µg/ml and 30.95 ± 1.97 µg/ml towards normal and irradiated fungal extract, respectively.