Abstract In this work, we evaluate the efficiency of biosurfactants produced by Bacillus subtilis LSFM-05 for the dispersion of acid-treated multi-walled carbon nanotubes (CNT-LQES1) and the effect of dispersion on toxicity testing with Daphnia similis. Carbon nanotubes are very hydrophobic materials and they readily agglomerate in mineral water. As a result, in order to determine their toxicity it is critical to evaluate methods to disperse these nanomaterials in a biologically compatible manner. The biosurfactant used in this work, termed BioS, which is a mixture of the lipopeptides (surfactin and fengycin), was found to be non-toxic to D. similis in an acute toxicity test (48h) and it was an excellent dispersing agent for CNT-LQES1 in reconstituted mineral water. Monitoring in real-time using the nanoparticle tracking analysis (NTA) showed that the colloidal stability of the CNT-LQES1 suspension dispersed with BioS was highly stable. These findings are encouraging for the application of biosurfactants as nontoxic dispersion agents in the emerging fields of bionanotechnology and nanotoxicology.