This research aimed to identify, isolate, and screen fungi from 10 soil samples. Using the agar well diffusion technique, the isolated cultures' capacity to generate antimicrobial metabolites was assessed against the virulent strains E. coli ATCC 8739, Staphylococcus aureus ATCC 6538, and Candida albicans ATCC 10231. Ten fungal isolates were crudely extracted, and the extract showed antibacterial action against one or more of the diseases under investigation. Further investigation was directed towards the NS6 isolate, which exhibited the highest level of activity among all the isolated strains against all tested pathogens. They matched them to other pertinent GenBank (NCBI) sequences using a particular internal transcribed spacer primer (ITS1/ITS4). Additionally, a variety of solvents were tested to determine which one produced the highest concentration of antibacterial compounds. PTLC and TLC bioautography have been used to purify antimicrobial substances. Using molecular identification, the most potent isolated fungus was identified as Aspergillus niger. Using IR, NMR, and mass spectra, the most promising bioactive molecule extracted from Aspergillus niger was discovered as (3-ethoxy-6-ethyle-8-hydroxy-4-p-tolyl-isochroman-1-one). The active compound extract of the Aspergillus niger strain was subjecte to column chromatography. Among the 12 fractions collected, the fraction number 4 had the highest overall antimicro bial activity with inhibition zone of 28 mm against Escherichia coli, 26 mm against Staphylococcus aureus and 25 mm against Candida albicans .