The present research work was undertaken with a mandate to study carbon spectrum utilization and structural characterization of biosurfactant produced by indigenous Pseudomonas aeruginosa NCIM 5514, which showed unique properties to utilize a large number of carbon sources effectively for production of biosurfactant, although glucose was the best carbon substrate. In Bushnell-Hass medium supplemented with glucose (1%, w/v), 3.178 +/- 0.071 g/l biosurfactant was produced by this isolate in 96 h. The biosurfactant produced showed surface tension and emulsification activity values from 29.14 +/- 0.05 to 62.29 +/- 0.13 mN/m and 88.50 +/- 1.96 to 15.40 +/- 0.91%, respectively. Toluene showed highest emulsification activity followed by kerosene. However, kerosene exhibited emulsion stability for 30 days. Biosurfactant was characterized as a mixture of di-rhamnolipid (Rha-Rha-C-10-C-14:1) and mono-rhamnolipid (Rha-C-8-C-10) by FTIR, ESI-MS and LC-MS techniques. High biosurfactant yield opens up doors for the isolate to find utility in various industries. (C) 2016 Elsevier Ltd. All rights reserved.