The antimicrobial properties of methyl (MTS) and ethyl (ETS) esters of thiosulfonic acid alone and in combination with rhamnolipid-biosurfactant (RL) have been characterized for their ability to disrupt the normal physiological functions of living pathogens. Bactericidal and fungicidal activities of MTS and ETS and their combination with rhamnolipid were demonstrated on strains of Pseudomonas aeruginosa, Bacillus subtilis, Alcaligenes faecalis, and Rhizopus ngtricans. It was found that the combination of rhamnolipid and thiosulfonic esters has a synergistic effect leading to decreasing of bactericidal and fungicidal concentrations of MTS and ETS. More extensively was studied the effect of rhamnolipid on the lipid composition of B. subtilis bacterial membrane. To our knowledge, in this article is reported for the first time a remarkable increase of negatively charged phospholipid cardiolipin in the presence of rhamnolipid. The capacity of RL as a surface-active substance was confirmed by scanning electron microscopy (SEM). The occurrence of surface infolds and blebs on B. subtilis shown by SEM, was not accompanied by changes in membrane permeability tested by a live/dead viability staining for fluorescence microscopy. When RL was applied in combination with MTS, a dramatic permeability shift for propidium iodide was observed in vegetative cells.