The DfrA1 protein provides trimethoprim resistance in bacteria, especially Klebsiella pneumoniae and Escherichia coli, by modifying dihydrofolate reductase, which reduces the binding efficacy of the antibiotic. This study identified inhibitors of the trimethoprim-resistant DfrA1 protein through high-throughput computational screening and optimization of 3,601 newly synthesized chemical compounds from the ChemDiv database, aiming to discover potential drug candidates targeting DfrA1 in K. pneumoniae and E. coli. Through this approach, we identified six promising DCs, labeled DC1 to DC6, as potential inhibitors of DfrA1. Each DC showed a strong ability to bind effectively to the DfrA1 protein and formed favorable chemical interactions at the binding sites. These interactions were comparable to those of Iclaprim, a well-known antibiotic effective against DfrA1. To confirm our findings, we explored how the promising DCs work at the molecular level, focusing on their thermodynamic properties. Additionally, molecular dynamics simulations confirmed the ability of these six DCs to effectively inhibit the DfrA1 protein. Our results showed that DC4 (an organofluorinated compound) and DC6 (a benzimidazole compound) exhibited potential efficacy against the DfrA1 protein than the control drug, particularly regarding stability, solvent-accessible surface area, solvent exposure, polarity, and binding site interactions, which influence their residence time and efficacy. Overall, findings of this study suggest that DC4 and DC6 have the potential to act as inhibitors against the DfrA1, offering promising prospects for the treatment and management of infections caused by trimethoprim-resistant K. pneumoniae and E. coli in both humans and animals. However, further in vitro validations are necessary.