Fungal secondary metabolites possess powerful bioactive compounds formed by fungi isolated from diverse sources. An isolated fungus from the sandy soil of Al Mudawara Mountain in El Fayum governorate was used in this study 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 by different organic solvents (petroleum ether, ethyl acetate, and chloroform) by order. The well diffusion method was used to examine the antibacterial effects of these extracts against various pathogenic microbes. 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. respectively. However, lower inhibitory activity was detected with ethyl acetate extract against HepG2 and PC-3 cells, respectively. Additionally, the antioxidant activity was tested using the DPPH free radical scavenging assay. 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. Gas chromatography-Mass Spectroscopy (GC-MS) analysis of the petroleum ether extract detected various important compounds such as Hexadecenoic acid, oleic acid, and 10,13-Octadecadiynoic acid. In conclusion, Aspergillus flavus ON764430 extract had moderate antimicrobial, antioxidant, and antitumor properties. To find out how the activities work, additional pharmacological and in vivo studies were suggested.