Environmental occurrence and hazardous nature of heterocyclic polyaromatic hydrocarbons (heterocyclic PAHs) has the potential to threaten the health of aquatic ecosystems. Here, we investigate the acute toxicity of heterocyclic PAHs (log K_OW 3.7–6.9) to aquatic organisms: marine bacteria (Aliivibrio fischeri), freshwater green algae (Raphidocelis subcapitata), and water fleas (Daphnia magna) using passive dosing to maintain stable exposure. The membrane-water partition coefficient (K_MW) of the heterocycles was measured to elucidate its relationship with toxicity. Our findings show that the tested heterocycles had little inhibitory effect on A. fischeri, while most compounds were highly toxic to R. subcapitata and D. magna. Toxicity generally increased with increasing K_MW values, and nonpolar narcosis was identified as the most likely mode of toxic action of the heterocycles. Comparison of standard protocols with passive dosing emphasizes the importance of maintaining a constant concentration during toxicity testing, as very high losses occurred in standard tests and passive dosing experiments revealed higher toxicities. These results indicate a potentially high risk to aquatic life and call for more in-depth investigation of the (eco)toxic effects of NSO-PAHs.