Benzo[a]pyrene (B[a]P) is a common environmental pollutant produced from the incomplete combustion of organic matter, such as fossil fuel emissions. In vertebrates, B[a]P exposure is associated with hyperactivity, though the underlying mechanisms driving this effect are undescribed. Mounting evidence indicates that the gut microbiome, which is the diverse community of microorganisms that comprise the gastrointestinal tract, could mediate the impact of B[a]P exposure on vertebrate behavior. In particular, an array of evidence raises the hypothesis that the gut microbiome’s metabolism of B[a]P influences its toxicity in a way that impacts B[a]P’s effect on early-life behavioral development in vertebrates. Alternatively, B[a]P could negatively alter the composition of the gut microbiome, inducing dysbiosis and impairing behavioral development. In this study, we explicitly tested these hypotheses using a zebrafish model. Zebrafish embryos colonized with conventional, conventionalized or germ-free microbiomes were exposed to varying concentrations of B[a]P and their photomotor response was assessed at 1 and 5 days post fertilization (dpf). Relative to conventionally reared zebrafish, germ-free zebrafish expressed a hyperactive photomotor response, and conventionalized expressed a hypoactive photomotor response in the dark following a light-dark transition at 5 dpf. These data suggest that presence or absence of the microbiome impacts behavioral development in juvenile zebrafish. This research builds on the body of evidence demonstrating the gut microbiome’s important role influencing pollutant metabolism and impact on vertebrate neurological development.