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Iron (Fe) and manganese (Mn) are metals commonly found at high concentrations in underground water. These metals are essential for the good functioning of living organisms, but high concentrations lead to imbalance, potentiating the appearance of pathologies. This study aimed to evaluate the effect of exposure to naturally occurring metals in groundwater, using zebrafish (Danio rerio) as an experimental model. Thus, zebrafish were exposed to Fe (0.8 and 1.3 mg/L), Mn (0.2 and 0.4 mg/L), and groundwater collected from deep tube wells with Fe and Mn (Fe 0.8/Mn 0.2 mg/L and Fe 1.3/Mn 0.4 mg/L) for 30 days. Bioaccumulation of these metals has been demonstrated in the livers and muscles of zebrafish. Acetylcholinesterase activity changed only in zebrafish muscles in all groups. Sulfhydryl levels changed mainly in the group Mn 0.4. SOD/CAT ratio decreased in the groups Fe 0.8 and 1.3, Mn 0.4, and Fe 0.8/Mn 0.4. An increase in the frequency of micronucleus in all groups was shown as a consequence of these changes. Behavioral parameters (time and distance traveled, mean speed, turn angle, latency, and number of crossings between compartments) have also changed, mainly in the groups Fe 1.3, Mn 0.4, and Fe 1.3/Mn 0.4. Therefore, long-term exposure to Fe and Mn, even at not so high concentrations, may cause biochemical, genotoxic, and behavioral changes in zebrafish.