The extract of Hops (Humulus lupulus L.), a herb medicine with a rich history spanning over 2000 years, has recently received extensive attention in food, health, and biomedicine. However, its reports in oral application are rare. To provide fundamental information for further clinical study in oral treatment, in this work, pleiotropic activity of hop extract including the stability, cytotoxicity, antioxidant activity, and antimicrobial activity of hop extract are investigated. The UV and thermal stability of hop extract were assessed using FT-IR analysis, while antioxidant activity was determined through experiments measuring DPPH, ABTS, and hydroxyl radical scavenging activities. Cytotoxicity was evaluated using the CCK-8 assay. Furthermore, we examined the antimicrobial activities of hop extract against four pathogenic microorganisms commonly associated with oral diseases—namely, E. faecalis, S. aureus, E. coli, and C. albicans—through inhibition zone and colony-forming unit assays. Experimental results show that hop extract has good UV and thermal stability, as well as good biological biocompatibility and antioxidant activity. Importantly, antimicrobial tests indicate that hop extract exhibits not only strong antibacterial activity against both Gram-positive and Gram-negative bacteria, but also effective antifungal ability. This study highlights the potential of hop extract as a promising antimicrobial agent for oral treatment. It serves as a crucial first step towards further applications and paves the way for the development of new treatments in oral therapies.