Environmental contaminants represent a poorly understood ecotoxicological and health risk. Here, we advanced a high-speed optical mapping (OM) technique to non-invasively track voltage dynamics in living zebrafish larvae’s heart and brain and investigate the effects of selected pesticides.OM allowed high resolution (∼17x) and fast acquisition (100 to 200 frames/s) of the voltage signal generated in the heart and brain after immersion of the zebrafish larvae in a voltage-sensitive dye. First, we used varying temperatures (20 °C to 25 °C) to test the adequacy of OM in capturing cardiac and brain voltage changes. Then, we tested the effects of glyphosate or a selected pesticide cocktail (2 to 120 h post-fertilization), accounting for their environmental thresholds and mimicking high-level exposure. Glyphosate (0.1 and 1000 μg/L) and the pesticide cocktail (0.1 and 10 μg/L) did not alter cardiac activity, except for a trend increase in heart rate variability at high glyphosate dose. Fourier transform (FT) analyses indicated that glyphosate reduced the abundance of low-amplitude voltage activities in the brain at the target low-frequency range of 0.2–15 Hz. The anatomical fragmentation of the brain into four regions, right and left diencephalon (RD and LD) and right and left optic tectum (ROT and LOT), confirmed the impact of glyphosate on the larvae brain and revealed a specific adaptation to the pesticide cocktail in the RD and ROT regions.In summary, OM captured heart and brain voltage changes in zebrafish larvae, with discrete patterns of brain depolarization in the presence of specific water contaminants. Here we discuss the relevance of these findings to ecotoxicology and exposome research.