Background: Infections caused by drug-resistant microorganisms have been increasing worldwide, thereby being one of the major causes of morbidity in the 21st century. Klebsiella pneumoniae is one such bacteria causing lung inflammation, lung injury and death. The emergence of hyper-virulent and drug-resistant species, such as ESBL and CRKP, has made this microbe a serious and urgent threat. The pace of emergence of these species is outgrowing the development of novel drug and vaccine candidates, thereby shifting the research focus towards the drug repurposing approach. Objective: The study aimed at Homology Modeling of Thymidylate Synthase, verification of modeled structure, Molecular Docking and Molecular Dynamic Simulation of the docked complex, and finally, in vitro analysis of 5-FU activity against Klebsiella pneumonia. Methods: The 3D structure of Thymidylate Synthase was predicted using the Swiss model server and validated by in silico approaches. The protein-protein interactions were determined using the STRING database. Molecular Docking has been carried out and MD simulations of 5-FU with the predicted structure of thymidylate synthase have been conducted. Finally, in vitro antimicrobial drug sensitivity assay has been carried out at different concentrations. Result: Hydrogen bond was observed in Molecular Docking, protein-ligand complex remained stable during simulation, while 5-FU showed antimicrobial activity against Klebsiella pneumonia during the in vitro study. Conclusion: Both in silico as well as in vitro analysis have indicated that 5-FU can potentially be developed as an antimicrobial agent towards Klebsiella pneumonia.