In-situ applications such as Microbial Enhanced Oil Recovery (MEOR) and remediation of contaminated sites demand production of biosurfactants in large quantities under oxygen limiting conditions. Few microorganisms have been isolated so far which can cater such need. In this paper, the characteristics of a biosurfactant produced under complete anaerobic conditions are presented. A novel biosurfactant producing microorganism, Pseudomonas sp ANBIOSURF-1 was isolated in our laboratory, from a microbial consortium enriched from municipal sewage sludge. The microorganism utilized vegetable oils and produced biosurfactant under complete anaerobic conditions. TLC tests revealed the presence of sugar and lipid as hydrophilic and hydrophobic moieties respectively. The biosurfactant synthesized under anaerobic condition thus belonged to class of glycolipids similar to that of rhamnolipids. The biosurfactant had a very low CMC value of 52 mg/l. The biosurfactant displayed good emulsifying activity over chlorinated solvents than the petroleum derivatives. The results suggest that Pseudomonas sp ANBIOSURF-1 could potentially be used for remediation of sites contaminated with chlorinated solvents through in-situ biosurfactant production. © (2010) Trans Tech Publications.