Soil is a primary resource used by mankind to ensure its needs mainly through agriculture. Its sustainability is regulated by the indigenous organisms it contains such as microorganisms. Current agricultural practices employ mixtures of pesticides to ensure the crops yield and can potentially impair these non-target organisms. However despite this environmental reality, studies dealing the susceptibility of microorganisms to pesticide mixtures are scarce. In this context, we designed a 3-month microcosm study to assess the ecotoxicity of realistic herbicide mixtures of formulated S-metolachlor (Dual Gold Safeneur®), mesotrione (Callisto®), and nicosulfuron (Milagro®) on the abundance, the diversity, and the activities of microorganisms from a “clay/organic matter-rich” soil, with a particular attention given to N-cycle communities. These communities appeared to be quite resistant to realistic mixtures even if transient effects occurred on the N-cycle-related communities with an increase of ammonification and an inhibition of nitrification as a short-term effect, followed by an increase of denitrification and an accumulation of nitrates. As nitrates are known to be highly leachable with a strong pollution potential, intensive studies should be carried out at field level to conclude on this potential accumulation and its consequences. Moreover, these data now need to be compared with other agricultural soils receiving these herbicide mixtures in order to bring general conclusion on such practices.