Fungal secondary metabolites possess powerful bioactive compounds formed by fungi isolated from diverse sources. In this study, a fungus was isolated from the sandy soil of Al Mudawara Mountain at El Fayum governorate to examine its antibacterial activity, cytotoxicity against cancer cells, and antioxidant effect. The fungus was identified using morphological and molecular methods as Aspergillus flavus, with a 99.29 percent similarity rate. The fungal secondary metabolites were extracted using different organic solvents (petroleum ether, ethyl acetate, and chloroform) in order. These extracts were tested for their antimicrobial activities against some pathogenic microorganisms using the well diffusion method. Furthermore, using the MTT viability assay, the antitumor properties of the active extracts were tested against HepG2 and PC-3, two different tumor cell lines. Aspergillus flavus petroleum ether extract had high inhibitory activity against HepG2 and PC-3 cells, according to the findings, However, lower inhibitory activity was detected with ethyl acetate extract against HepG2 and PC-3 cells, respectively. Also, the DPPH free radical scavenging assay was used to assess the antioxidant activity. The petroleum ether extract from Aspergillus flavus showed moderate antioxidant activity with IC50 of 272 ± 3.7µg/ml followed by ethyl acetate 901.3 ± 42.8µg/ml. GC-MS analysis of the petroleum ether extract showed the presence of different potent products. In conclusion, Aspergillus flavus ON764430 extracts made from ethyl acetate and petroleum ether had moderate antimicrobial, antioxidant, and antitumor properties. To find out how the activities work, additional pharmacological and in vivo studies were suggested.