The severity and perseverance of inflammation have been demonstrated in many health conditions. The limitations of existing medications suggest the need for new alternative anti-inflammatory medications. In our earlier studies, we demonstrated the topical anti-inflammatory potential of the crude ethanolic extract of Tetrastigma sulcatum leaves and its fractions. In the present study, we further explored the anti-inflammatory activity of T. sulcatum extract, fractions, pure compound and its derivatives using in vitro and in vivo bioassay techniques. We attempted to isolate a pure compound from the leaf extract and identified it as a Friedelan-3β-ol (CI). Furthermore, Friedelinol acetate (C II) and friedelinol methyl ether (C III), derivatives of Friedelan-3β-ol (CI) were synthesised. LPS-induced inflammatory RAW 264.7 macrophages were used as in vitro model to study anti-inflammatory and anti-oxidative effects. Inflammation-induced oxidative damage was found to be restricted significantly (P < 0.001), with scavenging activity and increased SOD activity of crude extract and fractions. Treatment with crude extract (TSETOH) and fractions (TSHEX, TSTOL) significantly reduced (P < 0.001) the mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and nitric oxide (NO) production in LPS-stimulated inflammation in RAW 264.7 cells in a dose-dependent manner. Likewise, compounds CI and CIII showed a similar pattern of significant inhibition (P < 0.001) of pro-inflammatory cytokines and NO production in a dose-dependent manner. An in vivo study in a carrageenan-induced mouse paw oedema model demonstrated reduced paw oedema and pro-inflammatory cytokines in a dose-dependent manner upon treatment with the extract, its fractions, pure compound (CI), and their derivatives (CII, and CIII). The present study confirmed the anti-inflammatory activity of T. sulcatum, suggesting that Friedelan-3β-ol is an active component of the crude extract.