Hypothesis: The cytotoxicity of biosurfactants on cell membranes may be influenced by composition of their hydrophilic head and hydrophobic tails. It is hypothesised that they form mixed micelles which exert a detergent-like effect that disrupts the plasma membrane. The functional physico-chemical and biocidal characteristics of four biosurfactants were concurrently investigated to determine which of their structural characteristics may be tuned for greater efficacy. Experiments: Rhamnolipid-95, rhamnolipid-90, surfactin and sophorolipid were characterised using FTIR, LC-MS, HPLC, surface tension and critical micelle concentration. Their biocidal activity against HEN 293, MCF-7 and THP-1 cell lines were investigated by MTT assay, using doxorubicin as cytotoxic control. Growth curves were established for all cell lines using trypan blue (TB) and MTT assays, corresponding doubling time (DT) and growth rate were obtained and compared. Findings: HEK 293 cell-line had the highest growth rate amongst the three cell lines. For TB assay, growth of HEN 293 > THP-1 and for MTT, HEN 293 > MCF-7 while the DT was in the order of THP-1 > MCF-7 > HEK 293. Sophorolipid showed anti-proliferative activity comparable to doxorubicin on THP-1 > MCF-7 > HEK 293. THP-1 showed high sensitivity to sophorolipid with IC50 of 10.50, 25.58 and 6.78 (mu g/ml) after 24, 48 and 72 h respectively. However, sophorolipid was cytotoxic from 24 to 72 h on HEK 293 cell lines with IC50 of 21.53, 40.57 and 27.53 mu g/ml respectively. Although, doxorubicin showed higher anti proliferative activity than all biosurfactants, it had poorer selectivity index for the same time durations compared to the biosurfactants. This indicates that biosurfactants were more effective for slowing the growth of the tested cancer cell lines and hence may be potential candidates for use in human cancer therapy. Physico-chemical characteristics of the biosurfactants suggest that their mechanism of action may be due to activity on the cell membrane. (C) 2016 Elsevier Inc. All rights reserved.