Abstract: To explore the taste characteristics and potential biological activities of the decapeptides of Stropharia rugosoannulata, two decapeptides (RIEDNLVIIR and SLPIKPRVPF) were selected to predict and validate the taste-presenting properties and ACE inhibitory activity mechanism by using virtual screening, molecular docking, and molecular interactions techniques. The results showed that the two decapeptides of S. rugosoannulata all had salty and umami tastes and ACE-inhibited peptide fragments. RIEDNLVIR had a salty taste, and SLPIKPRVPF had an umami taste. Two decapeptides of S. rugosoannulata could strongly bind to ACE receptors to form hydrogen bonds and electrostatic interactions. The in vitro activity validation results showed that the salty decapeptide RIEDNLVIIR inhibited the ACE well with an IC₅₀ value of 0.012 mg/mL. The molecular interaction thermodynamics and kinetics results showed that the binding between RIEDNLVIR and ACE receptor was the specific binding of enthalpy-driven reaction. The results of virtual screening activity prediction, in vitro activity validation, and molecular docking and molecular interactions for ACE inhibition mechanism analysis were consistent. The study provides a theoretical basis for understanding the taste characteristics and ACE inhibition mechanism of S. rugosoannulata decapeptides and lays a foundation for applying the decapeptides with ACE inhibitory activity in healthy condiments and functional products.