This study has investigated the antimicrobial potential of ring opening metathesis polymerization (ROMP)-based hydrogels. Initially, double charge containing cationic (DABCO)-ROMP polymers were synthesized and characterized. Subsequently, DABCO-ROMP polymer was added into a hydrogel formulation comprised of acrylamide (AAm), N,N'-methylenebis(acrylamide) (BAAm) and/or poly(ethylene glycol) diacrylate (PEG diacrylate, M-n = 700 g/mol) in different quantities to investigate the antimicrobial efficiency of the hydrogel formulations. N,N,N',N'-tetramethyl ethylene diamine (TEMED) is used with ammonium persulfate (APS) to catalyze polymerization in water. The structures of the resulting hydrogels were characterized using infrared spectroscopy (IR). The cross-linking properties and surface morphologies were investigated via the swelling behavior and scanning electron spectroscopy (SEM). Fluorescein (Na salt) staining measurements were also used to analyze the positive charge density of the hydrogels. The antimicrobial hydrogels showed excellent antimicrobial efficacy against Escherichia coli (E. coli, ATCC 25922), Staphylococcus aureus (S. aureus, ATCC 29213) and Candida albicans (C. albicans, ATCC 90028). It was observed that the cationic ROMP polymer in the hydrogel formulation played a crucial role in the biocidal activity. Furthermore, cytotoxicity studies on human umbilical vein endothelial cells (HUVEC) and tetrazolium salt assay (MTS) cell lines were also performed on the hydrogels and the most potent copolymer revealed no cytotoxicity. Therefore, the ROMP-based hydrogels show good biocidal properties with potential applications in various biomedical fields.