Fungi were isolated from Wadi El Rayan soils, and their secondary metabolites, extracted by petroleum ether, ethyl acetate, and chloroform, were tested for antimicrobial activities. Among the isolates, fungus-F1 extracts exhibited remarkable inhibitory activity against the tested microorganisms; therefore, it was subjected to morphological and molecular identification, and the fungus identified as Aspergillus fumigatus ON428521. With an inhibition zone ranging from 7mm to 21.4mm, ethyl acetate extract exerted the greatest activity. However, the petroleum ether extract only reached 16.5mm, and the chloroform extract demonstrated a lower activity level. Interestingly, activity was higher against tested Gram negative than those of Gram positive bacteria. Furthermore, Escherichia coli, Streptococcus mutans, and Cryptococcus neoformans were treated with ethyl acetate extract to explore possible effects on ultrastructure using transmission electron microscopy. The cell walls of treated cells lost their uniformity, ruptured, and became thicker. Moreover, the antitumor activities were evaluated against hepatocellular carcinoma (HepG2) and prostate carcinoma (PC-3) cell lines using the MTT assay, which revealed a high inhibitory activity with petroleum ether extract (IC50= 61.02 ± 1.64 and 90.31± 2.37μg/mL, respectively). Lower inhibition was detected with ethyl acetate extract (IC50 of 84.42±3.36 and 121.98 ±3.46μg/mL against HepG2 and PC-3, respectively). In addition, antioxidant activity was evaluated using the DPPH radical scavenging assay, and ethyl acetate extract demonstrated moderate activity with IC50 = 278.24 ± 8.52μg/mL, followed by petroleum ether (581.07 ± 41.95μg/mL). The phytochemical analysis showed that the ethyl acetate extract is rich in phenolics, followed by flavonoids and alkaloids while lipids are the major component of the petroleum ether extract. GC-MS analysis of petroleum ether extract gives dodecanamine, N, N-dimethyl-, lupeol acetate, amyrin, kojic acid, and oleanenol-acetate as the major components. In conclusion, ethyl acetate and petroleum ether extracts of A. fumigatus F1 exhibited moderate antimicrobial, antioxidant, and antitumor properties.