Nanoparticles are particles with at least one dimension between 1 and 100 nm. Due to their unique properties, gold nanoparticles (AuNPs) are used for drug delivery, diagnostics and cellular imaging, but there are concerns they interfere with neuronal development. In this study, we tested for potential neurotoxic effects by analyzing behavioral changes in zebrafish (Danio rerio) embryos and larvae exposed to AuNPs. We injected three different sizes of AuNPs (20, 40 and 80 nm) into either embryos at 2-4 hours post fertilization (hpf), or larvae, at 72 hpf. The AuNPs were delivered into the yolk sack of the embryos and the duct of Cuvier (i.e. the blood stream) in the larvae. Five concentrations of each AuNPs were tested in triplicate (1000, 500, 100, 50 and 5 µg/ml), all of which were below the lethal dose. Behavioral changes are a good indicator of neurotoxicity. Therefore, we used an automatic tracking system to analyze the distance travelled and time spent active in 96 hpf zebrafish. These endpoints were tested under a light-dark-light photo regime. In addition, we monitored motorneuron development using whole-mount immunohistochemistry staining for anti-acetylated tubulin (α-AT) at 48 hpf. We found that embryos injected with all three AuNP sizes showed reduced locomotor activity during the dark phase compared to controls, but there was no AuNP effect in the light phase. These reductions were concentration dependent only in 80 nm AuNPs, whereby the highest doses resulted in the lowest movement. We did not find any significant interaction of AuNP size on the reduction in locomotor activity. In contrast, we found no clear response patterns in larvae injected with AuNPs at 72hpf. Here, some concentrations increased, whereas others decreased, activity levels compared to controls during the dark phase. With respect to motorneuron development, we found larvae exposed to all sizes of AuNPs had shorter axons than controls. These results demonstrate that AuNPs have an effect on zebrafish behavior and axon development. As the behavioral effects were more apparent and consistent following the earlier exposure window, i.e. embryos, this suggests AuNPs interfere with the early stages of neurological development.