The aim of the present study was to investigate the excisional wound healing activity of a biosurfactant isolated from Bacillus stratosphericus sp. A15 using Catla catla fish fat as economic substrate. The organism was screened on the basis of blood haemolytic activity, cell surface hydrophobicity, oil displacement test and its promising capacity of reducing surface tension (ST). The biosurfactant was identified as surfactin lipopeptide using IR, 1H NMR and mass spectrometric analysis. Mass spectrometric data indicated the presence of amino acid sequence as Val/Asp/Val/Leu/Leu/GluOMe linked with β-hydroxy fatty acid moiety containing 14 carbons in normal, iso or anteiso forms. An ointment of the test biosurfactant enhanced the wound closure (97%) in 10 days compared to the untreated control group (72%) (P < 0.05). Histopathological study confirmed the healing effect of the test biosurfactant in terms of well developed keratinocyte, presence of hair follicles, vacuoles, higher number of intact cells in dermis layer and a thick epidermal layer. The biosurfactant also showed anti-oxidant and antibacterial activity against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 8739 which established its' additional advantage in wound protection. © The Royal Society of Chemistry.