A new amperometric biosensor based on inhibition of Pseudomonas putida has been developed to assess the acute toxicity of wastewater. This system uses the biological component immobilized on disposable screen-printed electrodes. The responses for a selected group of polar organic standard substances were studied using Pseudomonas putida as biological component. The results have been compared with responses obtained using the same system and Escherichia coli as biological component and with the bioluminescence inhibition of Vibrio fisheri using ToxAlert 100. Different properties, e.g. the standard deviation (SD) of the data, the goodness of fit (R2) and the standard deviation (Syx) of the vertical distances of the points from the inhibition curve, the 50% effective concentration (EC50) and the toxicity units (TII50) of the standard substance, were calculated and compared. This biosensor was used to assess the acute toxicity of real wastewater samples collected at different wastewater treatment plants (WWTP). Finally, a sequential solid-phase extraction (SSPE) procedure followed by liquid chromatography-mass spectrometry (LC-MS) was used to determine the polar organic toxic substances present in the wastewater samples.