There have been limited studies on the effects of toxicity modifying factors, such as dissolved organic matter (DOM), on the toxicity of metal mixtures to aquatic biota. Here we present the effects of DOM concentration (low: 2.8 ± 0.1 mg C/L; high: 11 ± 1.0 mg C/L) and DOM source (predominantly terrestrial or microbial) on the chronic toxicity of copper and nickel binary mixtures to the green freshwater microalga Chlorella sp. This was assessed by using a full factorial design of 72-h growth inhibition bioassays. Measured algal growth rate was compared with growth predicted by the concentration addition (CA) and independent action (IA) reference models. Model predictions were based on concentrations of dissolved metals, labile metals (measured by diffusive gradients in thin-films, DGT) and calculated free metal ions (determined by the Windermere Humic Aqueous Model, WHAM). Copper/nickel mixture toxicity was synergistic to Chlorella sp. in the absence of added DOM, with evidence of metal concentration-dependent toxicity at low effect concentrations. As DOM concentration increased, the mixture interaction changed from synergism to non-interaction or antagonism depending on the metal speciation method used. DOM source had no significant effect on mixture interaction when based on dissolved and free metal ion concentrations but was significantly different when based on DGT-labile metal concentrations. Ratio-dependent mixture interaction was observed in all treatments, with increased deviation from the reference model predictions as the mixture changed from nickel- to copper-dominated. This study demonstrated that both DOM concentration and source can significantly change metal mixture toxicity interactions and these interactions can be interpreted differently depending on the metal speciation method used.