Wastewater treatment plants are a major pathway for pharmaceuticals to the aquatic environment. Many pharmaceuticals, including non-steroidal anti-inflammatory drugs (NSAIDs), are chiral chemicals and the biological activity of their enantiomers can differ. Few studies have assessed the effects of different NSAID enantiomers on non-target organisms. However, this information is important for environmental risk assessment to ensure that the effects of more potent enantiomers are not overlooked. In the current study, enantiomers of naproxen, ibuprofen, ketoprofen and flurbiprofen were evaluated in bioassays with bacteria, algae and fish cells. All enantiomers induced bacterial toxicity, with (R)-naproxen more toxic than (S)-naproxen (EC50 0.75 vs 0.93 mg/L) and (S)-flurbiprofen more toxic than (R)-flurbiprofen (EC50 1.22 vs 2.13 mg/L). Both (R)-flurbiprofen and (S)-flurbiprofen induced photosystem II inhibition in green algae, with (R)-flurbiprofen having a greater effect in the assay after 24 h (EC10 5.47 vs 9.07 mg/L). Only the (R)-enantiomers of flurbiprofen and ketoprofen induced ethoxyresorufin-O-deethylase (EROD) activity in fish cells, while (S)-naproxen was 2.5 times more active than (R)-naproxen in the EROD assay. While enantiospecific differences were observed for all assays, the difference was less than an order of magnitude. This indicates that the risk of overlooking the effect of more potent NSAID enantiomers is minor for the studied test systems and supports the use of racemic (or single enantiomer) effect data for environmental risk assessment. However, further investigation of the (R)-enantiomer of commonly used NSAID ketoprofen is recommended as it was at least six times more potent in the EROD assay than the inactive (S)-ketoprofen.