<p>Soluble epoxide hydrolase (sEH) is a pivotal therapeutic target for inflammation management, as it regulates the hydrolysis of anti-inflammatory epoxy fatty acids (EpFAs). In this study, we investigated the sEH inhibitory potential of <i>Toxicodendron vernicifluum</i> (Stokes) F.A. Barkley heartwood. Bioactivity-guided fractionation of the ethanol extract identified the ethyl acetate-soluble fraction as the most active fraction (~ 60% inhibition at 50&#xa0;µg/mL). Subsequent purification yielded 11 polyphenols, all of which are known compounds previously reported from this species. While these are known phytochemicals, this study represents the first characterization of their inhibitory kinetics against sEH. Among the isolates, the aurone sulfuretin (<b>7</b>) and the flavonol fisetin (<b>5</b>) emerged as potent competitive inhibitors with IC₅₀ values of 8.8 ± 0.3 µM and 9.6 ± 0.8 µM, respectively. Conversely, the chalcone butein (<b>9</b>) exhibited a distinct non-competitive mode of inhibition (IC₅₀ = 21.4 ± 1.5 µM). Computational analyses, including molecular docking and 100 ns molecular dynamics (MD) simulations, elucidated the structural basis of these interactions. The simulations confirmed the stable engagement of sulfuretin within the catalytic pocket via hydrogen bonds with Asp335 and Tyr383, whereas docking studies indicated that butein occupies a peripheral allosteric site. These findings demonstrate that <i>T. vernicifluum</i> heartwood is a promising source of sEH inhibitors with dual mechanisms of action, highlighting its potential for development into anti-inflammatory therapeutics.</p>