© 2017 Elsevier B.V. The extensive use of 2,4-dichlorophenoxiacetic acid (2,4-D) in agriculture is an important source of pollution to water and soil. Toxicity of commonly used herbicides to non-target, planktonic photosynthetic organisms has not been described completely yet. Therefore, we determined the effect of subinhibitory 2,4-D concentrations on the Chlorophycean alga Ankistrodesmus falcatus and on a toxigenic strain of the cyanobacterium Microcystis aeruginosa. Population growth, photosynthetic pigments, macromolecular biomarkers (carbohydrates, lipids, and protein), and antioxidant enzymes (catalase [CAT], glutathione peroxidase [GPx], and superoxide dismutase [SOD]) were quantified, and the integrated biomarker response (IBR) was calculated. Scanning electron microscope (SEM) and transmission electron microscope (TEM) observations were also performed. The 96-h median inhibitory concentration (IC50) for 2,4-D was 1353.80 and 71.20 mg L− 1 for the alga and the cyanobacterium, respectively. Under 2,4-D stress, both organisms increased pigments and macromolecules concentration, modified the activity of all the evaluated enzymes, and exhibited ultrastructural alterations. M. aeruginosa also increased microcystins production, and A. falcatus showed external morphological alterations. The green alga was tolerant to high concentrations of the herbicide, whereas the cyanobacterium exhibited sensitivity comparable to other phytoplankters. Both organisms were tolerant to comparatively high concentrations of the herbicide; however, negative effects on the assessed biomarkers and cell morphology were significant. Moreover, stimulation of the production of cyanotoxins under chemical stress could increase the risk for the biota in aquatic environments, related to herbicides pollution in eutrophic freshwater ecosystems.