The yeast species viz. Rhodotorula muciliginosa and Candida rugosa isolated from hydrocarbon contaminated sites were capable of producing biosurfactants in the presence of 2{\%} (v/v) diesel as sole source of carbon and energy. The crude biosurfactants were purified using silica gel column chromatography followed by dialysis. Chemical structures of the purified biosurfactants were identified as diacetate acidic sophorolipid and mono acetate lactonic sophorolipid using FT-IR and GC-MS. The biosurfactants showed high physicochemical properties in terms of the surface activities when compared to synthetic surfactants, including Tween 80 and sodium dodecyl sulfate. Involvement of biosurfactant in physiological mechanism of diesel adsorption on yeast cell surface was characterized based on zeta potential measurement. When diesel oil was emulsified with biosurfactant, the surface charge of the diesel was modified resulting more adsorption of diesel on yeast cell surface. SEM analysis was used to monitor biosurfactants production by yeast species.