Surfactants (surface active agents) have a wide spectrum of use in the industry, but also in agriculture. Moreover, there is a tendency to use both synthetic (e.g. Tween 80) and natural (e.g. rhamnolipids) surfactants in bioremediation of contaminated sites. In this case, a potential toxicity of surfactants towards different elements of the ecosystem should be considered. The aim of this study was to determine phytotoxicity in relation to Sinapis alba (white mustard) seeds and toxicity towards activated sludge microorganisms of rhamnolipids and Tween 80 at different concentrations. The results indicate a higher phytotoxicity of rhamnolipids compared with Tween 80. Surfactants used at the highest concentration, (600 mgּ l-1) exhibit-ed an inhibition of roots growth by 69% and 16% (compared with a control) for rhamnolipids and Tween 80 respectively. The results of toxicity towards activated sludge microorganisms confirm the negative impact of both surfactants especially in high concentrations. In the two highest concentrations no significant difference between synthetic surfactants and biosur-factants was observed. The results suggest that rhamnolipids could not be considered as more ‘eco-friendly’ surfactants compared with synthetic Tween 80.