Materials that solidify in response to an initiation stimulus are currently utilized in several biomedical and surgical applications; however, their clinical adoption would be more widespread with improved physical properties and biocompatibility. One chemistry that is particularly promising is based on the thiol-ene addition reaction, a radical-mediated step-growth polymerization that is resistant to oxygen inhibition and thus is an excellent candidate for materials that polymerize upon exposure to aerobic conditions. Here, thiol-ene-based hydrogels are polymerized by exposing aqueous solutions of multi-functional thiol and allyl ether PEG monomers, in combination with enzymatic radical initiating systems, to air. An initiating system based on glucose oxidase, glucose, and Fe2+ is initially investigated where, in the presence of glucose, the glucose oxidase reduces oxygen to hydrogen peroxide which is then further reduced by Fe2+ to yield hydroxyl radicals capable of initiating thiol-ene polymerization. While this system is shown to effectively initiate polymerization after exposure to oxygen, the polymerization rate does not monotonically increase with raised Fe2+ concentration owing to inhibitory reactions that retard polymerization at higher Fe2+ concentrations. Conversely, replacing the Fe2+ with horseradish peroxidase affords an initiating system is that is not subject to the iron-mediated inhibitory reactions and enables increased polymerization rates to be attained.