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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-C₁₀-C_14:1) and mono-rhamnolipid …