The use of hydrogels in biomedical applications is widespread and constantly progressing, as the high water uptake affords tissue-like mechanical properties and facile functionality. Moreover, the gaseous molecule, nitric oxide (NO), has been shown to be responsible for various endogenous physiological functions including acting as a potent antimicrobial, released from macrophages during infection and wound healing processes. Consequently, the fabrication of hydrogel materials comprising synthetic NO donor molecules, such as S-nitrosothiols (RSNOs), reveals hydrogels with active functionalities such as enhanced antibacterial activity. However, hydrogel manipulation and fabrication techniques have consequences on NO release and donor stability. This dissertation aims to investigate the broad-spectrum antimicrobial efficacy of NO through a series of three projects, each with increasing complexity in aqueous and hydrogel systems. First, NO released from GSNO in aqueous solutions was tested against commercially available bacterial strains and clinically isolated drug-resistant bacteria strains of the same species. Similar killing capabilities demonstrate the clinical potential of such NO-releasing treatments. Second, the water-soluble NO donor, GSNO, was embedded into an alginate hydrogel, crosslinked into spherical beads and characterized for NO release, antibacterial capacity, and biocompatibility with mammalian cells. Lastly, GSNO in combination with a fluoride salt was incorporated into a co-system of alginate and Pluronic F-127 hydrogels. The composite system displayed potential for dual functional treatment of dental caries through bacterial killing and biofilm dispersion, and prevention of demineralization of a tooth enamel model. Taken together, the addition of GSNO to alginate hydrogels provides tunable NO release with the opportunity for further modifications specific to the biomedical application in question.