Protocol was developed for isolation, fermentation, purification and characterization of the 2-Hydroxygenistein antibacterial compound of endophytic fungi harbouring Indian medicinal plant Litsea glutinosa (Lour.) C.B. Rob. Endophytic fungal strain RP1 was identified as Phoma sp. by phenotypic characteristics as well as molecular sequencing. Screening of crude extract from Phoma sp. was investigated for their antibacterial activity. The optimum productivity of the antibacterial compound and biomass was achieved with optimized physico-chemical parameters containing Sabourauds dextrose broth (SDB), incubation period (14 days), temperature (27°C), pH (7), carbon source (maltose) and nitrogen source (peptone). The optimum carbon source and temperature for maximum antibacterial activity and biomass were peptone at 27°C respectively. Therefore, in the present invention it was revealed that there is no co-relation between biomass and antibacterial activity of the Phoma sp. RP1. Large scale production of the antibacterial metabolite was carried out in the bioreactor. All optimized physico-chemical parameters were applied for fermentation in the bioreactor. For purification of the antibacterial compound solvent-solvent extraction method was used. The n-butanol fraction of CFCF showed maximum antibacterial activity was further purified in column chromatography (hexane : n-butanol : bcetone in ratio 50:30:20) and a total 10 fraction (A-J) were eluted and observed their antibacterial activity. The fraction number E that showed maximum zone of inhibition against test bacteria was further purified by TLC in solvent mixture of petroleum ether and ethyl acetate (70:30) that gave three spot with R/values 0.26, 0.52 and 0.59 and fraction obtain having a R/value of 0.26 was giving the best antibacterial activity. TLC sample was again dissolved into the water: methanol (20:80) solvent system and chemical structure of the antibacterial compound was identified by LCMS/MS a
