The present study aims to synthesize and characterize two quaternary ammonium (QAM) based monomers such as - dimethyl-hexadecyl-methacryloxyethyl-ammonium iodide (DHMAI) and 2-dimethyl-2-dodecyl-1-methacryloxyethyl ammonium iodine (DDMAI) and assess their cytotoxicity and antimicrobial properties. The study also aims to incorporate the optimized concentration of these monomers as copolymerizing monomers into conventional Polymethyl methacrylate (PMMA) denture base resin and evaluate their suitability for prosthetic applications. DHMAI and DDMAI monomers were synthesized through a Menschutkin reaction and their chemical structure was characterized using FT-IR and 1H-NMR spectroscopy. Cytotoxicity was determined using Methyl Thiazolyl Tetrazolium (MTT) assay whereas antimicrobial activity was assessed using the agar-disc diffusion method. Subsequently, optimized concentrations of DHMAI or DDMAI, based on the cytotoxicity results, were added to conventional PMMA resin. Antimicrobial activity, cytotoxicity, surface hardness, and water sorption of PMMA denture base rein incorporated with DHMAI or DDMAI were evaluated. FT-IR and 1H-NMR results confirmed the structure of monomers and copolymerization of DHMAI and DDMAI with PMMA resin. DHMAI and DDMAI monomers were found to be cytocompatible with mouse fibroblast cells up to a concentration of 5 μg/mL and 20 μg/mL respectively. In addition, incorporation of DHMAI or DDMAI at 5 μg/mL and 20 μg/mL respectively into PMMA denture base material did not affect their cytocompatibility. PMMA denture base resin incorporated with DHMAI or DDMAI significantly reduced the adhesion of microbes. Further, an increase in the surface hardness and a reduction in the water sorption was observed. Hence DHMAI and DDMAI can be considered as potential candidates for imparting antimicrobial activity to polymeric denture base materials.