3,6,7-trimethyllumazine (Lepteridine™) is a newly discovered natural pteridine derivative unique to Mānuka () nectar and honey, with no previously reported biological activity. Pteridine derivative-based medicines, such as methotrexate, are used to treat auto-immune and inflammatory diseases, and Mānuka honey reportedly possesses anti-inflammatory properties and is used topically as a wound dressing. MMP-9 is a potential candidate protein target as it is upregulated in recalcitrant wounds and intestinal inflammation. Using gelatin zymography, 40 μg/mL Lepteridine inhibited the gelatinase activities of both pro- (22%, < 0.0001) and activated (59%, < 0.01) MMP-9 forms. By comparison, Lepteridine exerted modest (~10%) inhibition against a chromogenic peptide substrate and no effect against a fluorogenic peptide substrate. These findings suggest that Lepteridine may not interact within the catalytic domain of MMP-9 and exerts a negligible effect on the active site hydrolysis of small soluble peptide substrates. Instead, the findings implicate fibronectin II domain interactions by Lepteridine which impair gelatinase activity, possibly through perturbed tethering of MMP-9 to the gelatin matrix. Molecular modelling analyses were equivocal over interactions at the S1' pocket versus the fibronectin II domain, while molecular dynamic calculations indicated rapid exchange kinetics. No significant degradation of synthetic or natural Lepteridine in Mānuka honey occurred during simulated gastrointestinal digestion. MMP-9 regulates skin and gastrointestinal inflammatory responses and extracellular matrix remodelling. These results potentially implicate Lepteridine bioactivity in Mānuka honey's reported beneficial effects on wound healing via topical application and anti-inflammatory actions in gastrointestinal disorder models via oral consumption.