Introduction: Infectious diseases are affecting the world with their morbidity and mortality. Globally, more than one-third of the world population is infected with the bacteria that cause tuberculosis (TB) and each year approximately 9 million people affected with the disease and each year 2 million of those die. Aim: To synthesize a series of novel 3,4-dihydropyrimidine chalcones which have anti-mycobacterial activity and to perform docking studies for active compounds. Methods: A series of 6-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-one derivatives were synthesized and evaluated their antimycobacterial activity against M. tuberculosis H37Rv strain using Microplate Alamar Blue dye Assay (MABA). The docking calculations were done on protein, thymidylate kinase (PDB ID: 1G3U) employing in Genetic Optimization for Ligand Docking (GOLD v4.0.1 2008) software using Genetic algorithm. Results: Among all the synthesized derivatives, 5-(3-(2-thienyl) acryloyl)-6-methyl-4-phenyl-3, 4-dihydropyrimidin-2(1H)-one was exhibited potent activity compared with reference standards pyrazinamide and streptomycin. The potent compound forms H-bonding with Tyrosine39, Glutamic acid 166 and Arginine160 amino acids of the active site, a 2-thienaldehyde derivative which undoubtedly accounts for their superior activity associated with other analogs of the series. The docking results exposed useful information to understand the interaction mode between 3,4-dihydropyrimidine chalcone derivatives and thymidylate kinase will simplify the next cycle of drug design to reconnoitre the newer lead molecules. Conclusion: The interaction model between dihydropyrimidine chalcone derivatives and thymidylate kinase will facilitate the next cycle of drug design to explore the newer lead molecules.