Mango kernel oil (MKO), derived from mango kernels, considered to be one of the highly generated agro-industrial waste, is assessed for its use as substrate for sustainable production of rhamnolipids. In the present study, MKO in combination with glucose gave maximum rhamnolipid yield of 2.8 g/l which reduced the surface tension of water from 72 to 30 mN/m, holding a CMC of 80 mg/l and also showed high emulsification activity (73%) with diesel. Cell free broth was found to be stable even at high temperature (autoclaved at 121 degrees C for 30 min), pH value (up to pH 12) and salinity (up to 20% NaCl). The LC-MS data showed mono-rhamnolipid to be predominant congener followed by di-rhamnolipid in presence of MKO. Whereas, di-rhamnolipid was abundant when a combination of MKO with glucose was used. The produced rhamnolipid mixture showed good antifungal activity against various phytopathogens. (C) 2016 Elsevier Ltd. All rights reserved.