The evaluation of the ecotoxicological effects of water pollutants is performed by using different aquatic organisms. The effects of seven compounds belonging to a class of widespread contaminants, the benzo-fused nitrogen heterocycles, on a group of simple organisms employed in reference ISO tests on water quality (unicellular algae and luminescent bacteria) have been assessed to ascertain their suitability in revealing different contamination levels in the water, wastewater, and sediments samples. Representative compounds of benzotriazoles, benzothiazoles, and benzenesulfonamides, were tested at a concentration ranging from 0.01 to 100 mg L−1. In particular, our work was focused on the long-term effects, for which little information is up to now available. Species-specific sensitivity for any whole family of pollutants was not observed. On average, the strongest growth rate inhibition values were expressed by the freshwater Raphidocelis subcapitata and the marine Phaeodactylum tricornutum algae. R. subcapitata was the only organism for which growth was affected by most of the compounds at the lowest concentrations. The tests on the bioluminescent bacterium Vibrio fisheri gave completely different results, further underlining the need for an appropriate selection of the best biosensors to be employed in biotoxicological studies.