This study investigated the potential of oat avenanthramides (AVNs) to inhibit amyloglucosidase. Avenanthramide C (AVC, IC₅₀ 2.4 mg/mL) exhibited the strongest inhibition followed by Avenanthramide A (AVA, IC₅₀ 2.9 mg/mL) and Avenanthramide B (AVB, IC₅₀ 4.4 mg/mL). AVNs inhibit amyloglucosidase via a competitively dominant, mixed-type mechanism. This competitive character is mechanistically explained by AVN binding near the catalytic center, inducing a conformational change that reduces the active site volume and blocks substrate binding. Molecular dynamics simulations confirmed complex stability, with MMGBSA indicating superior binding affinity for AVC (-43.9 kcal/mol, AVB: -32.5 kcal/mol, AVA: -25.2 kcal/mol). Fluorescence quenching confirmed static binding and ligand-induced fluorescence shifts, indicating enzymatic conformational changes. Circular dichroism further revealed that AVNs induced structural alterations, exhibiting bidirectional effects by promoting unfolding in the lower temperature range while inhibiting it at elevated temperatures. These findings support oats as a source of natural amyloglucosidase inhibitors for functional foods aimed at glycemic control.