In the present study, Escherichia coli was successfully isolated from a catfish sample collected from the El-Moheet drain in Giza, Egypt, with a prevalence rate of 20.8%. The bacterial isolates were confirmed through biochemical tests, demonstrating characteristics consistent with E. coli, such as indole production, nitrate reduction, and acid production. The strain was further confirmed by 16S rRNA sequencing and deposited in GenBank (PQ218973.1). In parallel, five endophytic fungi were extracted from seagrasses in Wadi El-Natron, El-Beheira, Egypt, and exhibited notable antimicrobial properties. Crude extracts from these fungi, particularly the F2 isolate, showed strong antimicrobial activity, with an efficacy of 89.40 ± 2.44% against pathogenic microorganisms. The most potent endophyte was identified as Aspergillus sp < em>. through morphological and genetic analysis, classified as Aspergillus sp < em>. isolate NRC1, and deposited in GenBank (PQ270262.1). GC-MS analysis of the fungal extracts revealed 36 bioactive compounds, with trans-1,4-diacetoxy-2-butene being the most abundant. ADME analysis of this compound indicated favorable physicochemical properties, including high gastrointestinal absorption and blood-brain barrier permeability. It exhibited no significant inhibitory effects on major cytochrome P450 enzymes, suggesting a low risk of drug interactions. Despite potential challenges in synthetic optimization, trans-1,4-diacetoxy-2-butene shows promise for further therapeutic development.