Deep eutectic solvents (DESs) have been intensively investigated in recent years for their antibacterial properties, with DESs that comprise organic acids (OA‐DESs) showing promising antibacterial action. However, the literature is lacking a more in‐depth investigation that would cover the relative minimum inhibitory (MIC) and minimum bactericidal (MBC) values of these formulations, their mechanism‐of‐action, and concentration‐time‐dependent activity (TKA). In fact, a majority of the reports focused only on a few strains, which is not enough to determine the antibacterial potential of a substance. To bridge this gap, the antibacterial activity of classical DESs and OA‐DESs was assessed on 12 Gram‐negative and Gram‐positive bacteria strains, with some of them exhibiting specific resistance towards antibiotics. The investigated formulations of OA‐DESs comprised glycolic, malic, malonic, and oxalic acids as representatives of this group. Using a range of microbiological assays as well as physicochemical characterization methods, a major difference of the effectiveness between the two groups was demonstrated, with OA‐DESs exhibiting, as expected, greater antibacterial effectiveness than classical DESs. Most interestingly, slight differences in the MIC/MBC values as well as TKA profiles were observed between Gram‐positive and Gram‐negative strains. Transmission electron microscopy (TEM) analysis revealed the effect of the treatment of the bacteria with the representatives of the both groups of DESs, which allowed us to better understand the possible mechanism‐of‐action of these novel materials. This article is protected by copyright. All rights reserved