Thiophene is an important class of heterocyclic compound, has been shown to exhibit diverse biological and pharmacological activities such as anti-cancer, antioxidant, anti-inflammatory, antimicrobial, etc. In this study, a series of novel 2-Methyl-5-thiophen-7-(aryl-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one derivatives have been synthesized. All the synthesized compounds have been characterized by using elemental analysis, FT-IR, 1H NMR, 13C NMR spectroscopy and further supported by mass spectroscopy. Purity of all the compounds has been checked on thin layer chromatographic plate and HPLC technique. All the synthesized compounds were tested for their antibacterial and antifungal activity (MIC) in vitro by broth dilution method with two Gram-positive bacteria, two Gram-negative bacteria and two fungal strains. The biological activities of the synthesized compounds have been compared with standard drugs Ampicillin and Greseofulvin..The compounds exhibited significant antibacterial and moderate antifungal activities. These compounds can be further exploited to get the potent lead compounds. The detailed synthesis and the antimicrobial screening of the new compounds are reported.