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The toxic effects of mercury in earthworms and the potential alleviation effect of hydrogen-rich water (HRW) using ATR-FTIR and LC–MS analysis methods were investigated. Different concentrations of mercury chloride (H1: 5 µg/mL, H2: 10 µg/mL, H3: 20 µg/mL, H4: 40 µg/mL, and C1: control) and mercury chloride prepared in hydrogen-rich water (H5: 5 µg/mL, H6: 10 µg/mL, H7: 20 µg/mL, H8: 40 µg/mL, and C2: control) were injected into earthworms. The changes and reductions in some bands representing proteins, lipids, and polysaccharides (3280 cm−1, 2922 cm−1, 2855 cm−1, 1170 cm−1, and 1047 cm−1) showed that protective effects could occur in groups prepared with hydrogen-rich water. In the FTIR results, it was found that these bands in the H3 group were more affected and decreased by the influence of mercury on earthworms than the H7 group prepared with hydrogen. LC–MS analysis showed that the changes in some ions of the highest dose groups (H4 and H8) were different, and mercury caused oxidative DNA damage in earthworms. When the high-level application groups of mercury, i.e., H4 and H8 were compared with the controls, the ion exchange ([M + H] + ; m/z 283.1) representing the 8-Oxo-dG level in earthworms was higher in the H4 group than the H8 group. This reveals that HRW exhibited the potential ability to alleviate the toxic effects of mercury; however, a longer period of HRW treatment may be necessary to distinguish an obvious effect. The ATR-FTIR spectroscopy provided a rapid and precise method for monitoring the changes in biological tissues caused by a toxic compound at the molecular level.