<![CDATA[Endophytic fungi have emerged as promising sources of bioactive secondary metabolites for pharmaceutical discovery due to their ability to produce metabolites similar to those of their host plants. This study investigated the antibacterial potential of strain CATD-09, an endophytic fungus isolated from Cnidoscolus aconitifolius (Mill.) I.M. Johnst., through antibacterial evaluation, chemical characterization, and molecular docking analysis. The antibacterial activity of fungal fractions was assessed against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538 using the minimum inhibitory concentration (MIC) method. The most active fraction was subsequently characterized by Gas Chromatography–Mass Spectrometry (GC–MS) to identify its major constituents. To predict the possible antibacterial mechanism, the identified compounds were subjected to molecular docking against penicillin-binding protein 2a (PBP2a), a validated molecular target associated with β-lactam resistance. Fraction 1 exhibited moderate antibacterial activity against S. aureus ATCC 6538 with an MIC value of 128 μg/mL and stronger activity against E. coli ATCC 8739 with an MIC value of 64 μg/mL. GC–MS analysis identified 9,12-octadecadienoic acid (Z,Z)- as the predominant constituent, accounting for 44.47% of the total composition. Molecular docking demonstrated that this compound exhibited a stronger predicted binding affinity toward PBP2a than the reference ligand, indicating its potential to inhibit bacterial cell wall biosynthesis. Overall, these findings suggest that Alternaria sp. CATD-09 is a promising source of antibacterial metabolites and provide a scientific basis for further investigation of its bioactive compounds as potential lead candidates for antibacterial drug discovery.]]>
