Introduction: Due to the restrictive use of critical antibiotics by veterinarians in Europe, the use of first-line antibiotics such as the combination of sulfonamides (S) and trimethoprim (TMP) is likely to increase. The usual S:TMP formulations used in almost all veterinarian products on the market aims to produce an in vivo ratio of plasma concentration of 20:1, which was determined as the most effective 40 years ago. However, it is not always reached in the different animal species due to PK differences between sulfonamides and animal species. Therefore, it is necessary to determine the optimal range of S:TMP ratio to characterize properly the extent of the synergy for each sulfonamide. Objectives: The aim of this study is to determine the most synergistic ratio of three S/TMP combinations widely used in veterinary medicine: sulfamethoxazole (SMX)/TMP, sulfadimethoxine (SDMX)/TMP and sulfadiazine (SDZ)/TMP on several Escherichia coli strains (n = 9) with various susceptibility profiles to these drugs. Materials and Methods: Different ratios of SMX/TMP, SDMX/TMP and SDZ/TMP combinations were tested over a range of S and TMP concentrations (relevant with in vivo PK profiles), using checkerboard assay in conventional media. The Fractional Inhibitory Concentration Index (FICI) was calculated to identify the most synergistic ratios (FICI < 0.5) which are used in time-kill assays (fixed antibiotic concentrations) to document the time-effect and the concentration-effect relationships in a more mechanistic way. Results: For all studied strains, Minimal Inhibitory Concentrations (MIC) are lower when sulfonamides are associated with TMP compared to the MIC with sulfonamides alone, highlighting the synergism of these associations on Escherichia coli. The potency of this synergism appears to depend on the sulfonamide used in the combination as highest synergisms were found within TMP/S ratios ranging from 1/32 to 1/320; 1/64 to 1/4096 and 1/16 to 1/1280 for SMX, SDMX and SDZ respectively. Interestingly, for each strain the most synergistic ratios seems to be very close to the ratio of the MIC of the drugs used alone. Time-kill assays will allow us to better characterize the time-effect (currently ongoing). Conclusion: All these preliminary data on the antibacterial activity of optimal SMX/TMP, SDMX/TMP and SDZ/TMP ratios will generate rich PD data on the S:TMP combinations which will allow, in combination with PK profiles, the development of robust PK/PD models. The same work is ongoing with other relevant veterinary pathogenic bacteria.