With the increasing prevalence of allergic diseases, the need for new treatments has become more urgent. Among various interleukins, IL-33 plays a crucial role in the early stages of allergic responses. Therefore, inhibiting IL-33 could lead to the development of a novel drug with minimal side effects. Fraxinus rhynchophylla Hance (Oleaceae) is a traditional medicinal plant widely used in Asia, particularly its stem bark, which has been utilized in Korean folk medicine for its antibacterial, analgesic, and anti-inflammatory properties. In our study, the 80% methanol extract exhibited significant IL-33 inhibitory activity. To identify its bioactive constituents, the extract was fractionated using n-hexane, ethyl acetate (EtOAc), aqueous butanol, and water, and the inhibitory activity of each fraction was assessed. Since the EtOAc fraction showed the strongest activity, it was selected for further isolation. Six compounds (1–6) were isolated and their structures were elucidated by NMR and LC-MS analysis. The IL-33 inhibitory activity of the isolated compounds was evaluated, and molecular docking simulations were performed to support the in vitro findings. These findings suggest that secondary metabolites from F. rhynchophylla have potential as IL-33 inhibitors. This study was supported by the National Research Foundation of Korea grants funded by the Korean Government (NRF-2019R1A6A1A03031807 and NRF-2021R1A2C1093814) and the MSIT (Ministry of Science and ICT).