A library of 24 pyridinium-, imidazolium-, and cholinium-based ionic liquids (ILs) with varying alkyl chain from C2 to C16 was toxicologically profiled using naturally luminescent marine bacteria Vibrio fischeri. The toxicity (30-min EC50) of studied ILs to Vibrio fischeri ranged from 7.82 µM (4.2 mg/L) (PyC12Phe) to 3096 µM (1227 mg/L) (ImidC2Phe), i.e. from “toxic” (EC50 1–10 mg/L) to “not harmful” (EC50 > 100 mg/L). Inhibition of the bacterial luminescence upon 30-min exposure to ILs correlated well with bacterial viability (exposure for 4 h). The toxicity of studied ILs was largely driven by the length of the alkyl chain (hydrophobicity) and not the type of cationic part of the IL: starting from C10 all the ILs irrespective of the cationic part proved “toxic”. The toxicity of the studied ILs was increasing in parallel to their hydrophobicity up to log Kow = 1 (C8–C10) and then levelling up, being consistent with the previously obtained analogous data sets. The “cut-off” effect reported in this study for longer chain length members of the ILs series leads to the “limit” toxicity level for this type of ILs to be ca. 8 mM. Two open-access online tools (www.molinspiration.com and www.vcclab.org) have been applied for the calculation of the Kow values for the 24 ILs reported in this study and 21 ILs reported in the literature. This lead to plotting two nonlinear monotonic correlations between the values of experimental log (1/EC50) and calculated log Kow. The limitation of the online tools and an effect of the ILs structure on the “cut-off” effect have been discussed. The challenge of developing low microbial toxicity surface active ILs remains a significant task to overcome. Our results shed light on the new approaches for designing environmentally benign ILs and functional surfactants. As the hydrophobicity of the ILs significantly correlated with the toxicity, the Vibrio fischeri assay could be considered a powerful tool in providing toxicity data for building and evaluating the QSAR toxicity models for ILs. Display omitted •24 L-phenylalanine derived ILs were screened for toxicity using bacteria V. fischeri.•The toxicity of 24 ILs to V. fischeri ranged from toxic to not harmful.•Adverse effects of ILs depended on the length of the alkyl chain.•Bacterial light output inhibition was more sensitive endpoint than viability.•V. fischeri test allows toxicity screening of big panels of ILs for QSAR analysis.