There is a growing concern for the potential toxicity of engineered nanomaterials that have made their way into virtually all novel applications in the electronics, healthcare, cosmetics, technology, and engineering industries, and in particular, biomedical products. However, the potential toxicity of carbon 60 (C60) at the behavioral level has not been properly evaluated. In this study, we used idTracker, a multitracking algorithm to quantitatively assess behavioral toxicity induced by C60 nanoparticles (C60 NPs) in adult zebrafish. We demonstrated that locomotion, novel tank exploration, aggression, shoaling, and color preference activities of the C60 NPs-treated fish was significantly reduced. In addition, the C60 NPs-treated fish also displayed dysregulation of the circadian rhythm by showing lower locomotion activities in both day and night cycles. The biochemical results showed that C60 NPs exposure at low concentration induced oxidative stress and DNA damage, reduced anti-oxidative capacity and ATP (adenosine triphosphate) levels, and induced stress-associated hormones, hypoxia, as well as inflammation marker upregulation in muscle and gill tissues. Together, this work, for the first time, provide direct evidence showing that the chronic exposure of C60 NPs induced multiple behavioral abnormalities in adult zebrafish. Our findings suggest that the ecotoxicity of C60 NPs towards aquatic vertebrates should be carefully evaluated.