Chemicals considered as neuroactive (such as certain pesticides, pharmaceuticals, and industrial chemicals) are among the largest groups of bioactive substances recently detected in European rivers. However, the determination of nervous-system-specific effects has been limited using in vitro tests or conventional end points including lethality. Thus, neurobehavioral tests using in vivo models (e.g., zebrafish embryo) have been proposed as complementary approaches. To investigate the specificity and sensitivity of a light-dark transition locomotor response (LMR) test in 4 to 5 days post fertilization zebrafish with respect to different modes of action (MoAs), we analyzed a set of 18 environmentally relevant compounds with various anticipated MoAs. We found that exposure-induced behavioral alterations were reproducible and dependent on concentration and time. Comparative and quantitative analyses of the obtained locomotor patterns revealed that behavioral effects were not restricted to compounds primarily known to target the nervous system. A clear distinction of MoAs based on locomotor patterns was not possible for most compounds. Furthermore, chemicals with an anticipated same MoA did not necessarily provoke similar behavioral phenotypes. Finally, we determined an increased sensitivity (≥10-fold) compared to observed mortality in the LMR assay for five of eight neuroactive chemicals as opposed to non-neuroactive compounds.