Pesticides used in viticulture create a potential risk for the aquatic environment due to drift during application, runoff and soil leaching. The toxicity of sixteen pesticides and one metabolite were evaluated on the growth of two marine microalgae, Tisochrysis lutea and Skeletonema marinoi, in 96-h exposure assays conducted in microplates. For each substance, concentrations of stock solutions were analytically measured and abiotic assays were performed to evaluate the chemical stability of pesticides in microplates. For two chemicals, microalgae exposures were run simultaneously in microplates and culture flasks to compare EC50 calculated from the two exposure systems. Results from chemical analyses demonstrated the low stability of hydrophobic pesticides (log KOW > 3). For such chemicals, EC50 values calculated using measured pesticide concentrations were two-fold lower than those first estimated using nominal concentrations. Photosystem II inhibitors were the most toxic herbicides, with EC50 values below 10 μg L−1 for diuron and around double this for isoproturon. Chlorpyrifos-methyl was the only insecticide to significantly affect the growth of T. lutea, with an EC50 around 400 μg L−1. All fungicides tested were significantly toxic to both species: strobilurins showed low overall toxicity, with EC50 values around 400 μg L−1, whereas quinoxyfen, and spiroxamine, showed high toxicity to both species, especially to T. lutea, with an EC50 below 1 μg L−1 measured for spiroxamine in culture flasks. This study highlights the need to perform chemical analyses for reliable toxicity assessment and discusses the advantages and disadvantages of using microplates as a toxicity screening tool.