Seventy six bacterial and fungal strains were isolated from different oil contaminated soil and fruits from the region of southern Punjab. Isolation was carried by serial dilution method in YPSA medium and isolates were screened for biosurfactant production that was carried out by various methods including detection of halo on CTAB-MB agar plate, tilted glass test and oil spread assay. Eight of the high yielding strains (?30 g/l of glycolipids) namely Aspergillus niger EH6o, Aspergillus fumigatus SS6da1, Aspergillus flavus MTK7Dbii, Aspergillus oryzae EH6A-II, Enterbactersp SS7f3a, Alternaria alternate MtkC1 and Penicillium sp KEmalta, Candida albicans SSGbrii.The highest surfactant producing strain was identified by culturing the selected strains on different nutrient media, temperatures and pH. We are reporting hereby for the first time the glycolipid producing fungal and bacterial isolates of southern Punjab. . The strain was found to produce surfactants with excellent oil displacement and emulsification ability that are important for several industrial uses.