A hydrogel is a hydrophilic cross-linked polymer network which can contain a large amount of water. Hydrogels with distinguished interfacial physical toughness were analyzed for their potential application as antifouling coating materials, utilizing sum frequency generation (SFG) spectroscopy as the interfacial analytical technique. The surface structures of one sulfobetaine (SBMA) zwitterionic hydrogel (ZWHG) and two polysaccharide hydrogels (PHGs) were probed in air; their interfacial structures with silica were examined using SFG in water and protein solutions, respectively. Both ZWHG and PHGs interfaces in water were dominated by strongly hydrogen-bonded water molecules, but the bonding strength associated with ZWHG was much stronger. Although all hydrogels experienced interfacial change in the presence of protein solutions, after cleaning, the zwitterionic hydrogel interface recovered almost completely while the other two hydrogels were subject to irreversible protein adsorption. Additionally, orientational analysis of ZWHG methyl groups in water was conducted and related to the superior hydrogen-bonding strength of water molecules at the ZWHG interface. The interfacial structures of hydrogel materials probed by SFG can be correlated to their antifouling properties. This research highlighted the critical role that hydrogen-bonding strength of interfacial water molecules play for antifouling applications.