The growing popularity of zebrafish in psychopharmacology and behavioral brain research is partly due to the practicality and simplicity of drug administration in this species. Several drugs may be administered to zebrafish by immersing the fish in the drug solution. Furthermore, numerous drugs developed for mammals, including humans, have been found to show a similar effect profile in the zebrafish. Thus, the zebrafish has been suggested as a potentially useful animal screening tool. Despite decades of drug development, anxiety still represents a major unmet medical need, and the search for anxiolytic compounds is continuing. The zebrafish has been proposed for high throughput screens for anxiolytic compounds, and the effects of anxiolytic compounds on the behavior of zebrafish have started to be explored. Diazepam (Valium®) is a frequently prescribed human anxiolytic, a GABAA receptor agonist, has also started to be tested in zebrafish, but with occasional contradicting results. Here, we investigate the effects of diazepam in larval (6-day post-fertilization old) zebrafish in a black-white preference paradigm. We found significant white preference and thigmotaxis (edge preference) in our control fish, anxiety-like responses that habituated over time. However, unexpectedly, we observed no anxiolytic effects of diazepam on these behaviours, and only detected significant motor activity reducing effect of the drug. We discuss the complex interpretation of light/dark tests in zebrafish, and also speculate about the possibility of differential GABAergic mechanisms that diazepam affects in larval vs adult zebrafish.