Fungal endophytes are considered as repository of potent novel bioactive compounds with profuse chemical diversity. These bioactive compounds have been widely utilized in pharmaceutical and agrochemical industries. The present work was undertaken to bio-prospect the fungal endophytes from Rauwolfia serpentina, a medicinal plant used in multiple indigenous preparations, for possible therapeutic interventions. 21 fungal endophytes were isolated from Rauwolfia serpentina with maximum colonization in leaf (42.8 %) followed by bark (33.3%) and stem (23.8%). In the enzymatic activity screening, #15RSBNEY was found to be potent amylase producer and #3RSSTNEY as potential cellulase producer. Further, three isolates viz. #16RSLBRT, #10RSLBRT and #23RSSTNEY exhibited promising antibacterial activity with maximum activity exhibited by #10RSLBRT against all test strains of Staphylococcus aureus. Interestingly, it was observed that antibacterial activity of #10RSLBRT increased by two fold after fractionation with acetone and chloroform. Based on above result, the culture filtrate of #10RSLBRT was extracted with hexane, chloroform and ethyl acetate followed by agar well gel diffusion method for antibacterial activity. The crude ethyl acetate residue of #10RSLBRT hereby exhibited potent antibacterial activity. The potential isolate i.e. #10RSLBRT was identified by molecular taxonomic tools using internal transcribed spacer rDNA analysis and was clustered in the Fusarium incarnatum-equiseti complex. Further studies on the characterization of bioactive compounds of ethyl acetate extract of # 10RSLBRT are warranted for the development of anti-staphylococcal therapy.