In freshwater environments, microorganisms such as microalgae are influenced by the concentrations of dissolved chemicals but can modify the fate of these substances by biosorption, accumulation and even metabolization. In this laboratory study, we assessed the growth and physiology of non-axenic cultures of the chlorophyte Sphaerellopsis sp. exposed to environmental concentrations of diuron, irgarol and S-metolachlor (0.5, 0.5 and 5 μg·L−1, respectively) singly and in mixture, in the presence or absence of natural dissolved organic matter (DOM). The growth, photosynthetic efficiency and relative intracellular lipid content of Sphaerellopsis sp., as were measured after 14 days of exposure, as were the concentrations of bacteria in the cultures. DOM absorbance and fluorescence, and concentrations of the herbicides and their metabolites in the culture medium were also recorded. The growth of Sphaerellopsis sp. was very low in the absence of DOM but dramatically enhanced in treatments where DOM was added. As a result, the toxicity of the herbicides observed in treatments without DOM was overcome in those where DOM was added. The chemical characteristics of DOM were modified by the microalgae, and the fate of the herbicides was affected by the interaction between microorganisms (both bacteria and algae) and the DOM. Herbicide concentrations decreased over time, with a simultaneous increase in some of their metabolites, suggesting a biological degradation in the presence of DOM.