Objective:To isolate and characterize the bioactive secondary metabolite fromCallyspongia spp. associated fungi.Methods:In vitro antibacterial screening of fungi associated with Callyspongia species, collected from south east coast of India, against selected clinical isolates of bacteria were conducted in this study. The extracts showing good antimicrobial activity were subjected to further analysis to identify the active constituents sponge associated fungi (both biomass and filtrate) with five different solvents. The compound responsible for bioactivity was characterized using Fouvier-transform infrared (FT-IR) and gas chromatography-mass spectrometry(GC-MS) instrumental analysis to identify the functional group and compound. The molecular characterization of the elite fungal strains were done by isolating their genomicDNA and amplify the intal transcribed spacer(ITS) region of5.8srRNA using specific ITS primer. The novelty of the strain was proved by BlastN analysis against non-redundant(NR) database and hence was submitted to GenBank.Results: Active compound was Desmethylnomifensine confirmed byGC-MS and the potent fungi wasAspergillus flavusGU815344.Conclusions:The isolate exhibits a marked antagonistic activity against potential bacterial pathogens thus illuminating the advanced researches in this decade to focus on clinical pharmacology to identify novel therapeutic targets. The present study depicts a promising scenario to focus onAspergillus flavus derived compounds which can be easily scaled up for large biomass production and stable formulation as a drug.