Dihydrofolate reductase (DHFR) is a critical enzyme for bacterial survival, catalyzing the reduction of folate to tetrahydrofolate (THF), a vital cofactor for DNA synthesis. The significant structural differences between bacterial and human DHFR present an opportunity for targeted drug discovery, especially in combating antibiotic resistance. This study employed virtual screening and molecular dynamics simulations to identify novel inhibitors of DHFR in Klebsiella pneumoniae. Three ligands, specifically 5, 16, and 25, were found to exhibit strong binding affinities within the folate-binding site, effectively competing with the natural substrate. Detailed analyses revealed that ligand 5 engages critical residues such as Met20 and Asp27, indicating a potential mechanism for competitive inhibition. Ligands 16 and 25 also demonstrated substantial interaction stability, disrupting substrate binding through diverse intermolecular forces. Molecular dynamics simulations indicated consistent stability metrics across root-mean-square deviation (RMSD), polar surface area (PSA), and solvent-accessible surface area (SASA) assessments, confirming robust binding characteristics. These findings suggest that ligands 5, 16, and 25 represent promising leads for the development of novel antibacterial agents targeting DHFR, with the potential to circumvent common resistance mechanisms. Future research will be essential to validate these compounds in vitro and in vivo, paving the way for optimized therapeutic options against multidrug-resistant pathogens.