Previous studies have shown that contaminant toxicity to target organisms is altered by the presence of dissolved organic matter (DOM). Contaminants can bind to DOM and this may alter the bioavailability and subsequent toxicity of the contaminants. However, molecular-level techniques are needed to more closely evaluate the impact of DOM on the sub-lethal biochemical responses to emerging contaminants. To investigate how DOM may alter the metabolic response to organic contaminant exposure, 1H nuclear magnetic resonance (NMR)-based metabolomics was used to investigate how the metabolome of Daphnia magna changes when Suwannee River DOM (5 mg organic carbon/L) is included in the acute exposure of four contaminants with varying hydrophobicity. Sub-lethal concentrations of the hydrophobic contaminant 17α-ethynylestradiol (EE2), the relatively more polar compounds carbamazepine and imidacloprid, or the anionic contaminant perfluorooctane sulfonate (PFOS) were used. A 48-h exposure to DOM alone had a minor impact on the metabolome of D. magna. There were significant increases in amino acids from EE2 exposure which were reduced in the presence of DOM, suggesting that DOM may alleviate the sub-lethal metabolic response from EE2 exposure through sorption and a reduction in freely dissolved EE2. The metabolome was relatively unaltered with exposure to carbamazepine and imidacloprid in the presence of DOM which is likely because these contaminants are water soluble and did not strongly interact with DOM. PFOS exposure resulted in a more significant metabolic response with DOM suggesting that DOM enhanced the uptake and bioavailability of PFOS in D. magna. As such, the presence of DOM should be considered when determining sensitive molecular-level changes in organisms to sub-lethal organic contaminant exposure.