Glyphosate is a widely-used herbicide that is frequently found as a pollutant of soil and water runoffs. Glyphosate toxicity is controversial but a toxic synergy with other molecules could result in deleterious consequences for living organisms and for the human health. Using budding yeast (Saccharomyces cerevisiae) as a eukaryotic model organism, we report here a strong toxic synergy between glyphosate and 2,4-dinitrophenol (DNP), a phenolic compound derived from diesel engine’s combustion and industrial pollutant found frequently in surface water and rainfall. Glyphosate concentrations below 600 mg/L did not affect yeast growth but exhibit dose-dependent toxicity in the presence of non-toxic DNP concentrations (below 1 mM). This so-called ‘cocktail effect’ increases with DNP concentration. Yeast growth is totally abolished in the presence of the highest concentration of both molecules. We explored the implication of oxidative stress in this synergistic effect of glyphosate and DNP, by measuring H2O2 concentrations in the culture media, and by comparing cta1Δ/ctt1Δ catalase double-mutant with wild-type yeast. We did not find any glyphosate-DNP enhanced susceptibility for the catalase mutant and did not observe any clear increase of H2O2 in the presence of the pollutant mixture. All these data suggest that the redox homeostasis is not involved in this toxic synergy, that remains to be explained.