Display omitted •Nonapeptides and decapeptides were the main active peptides prepared by probe ultrasound.•Heptapeptides were the main active peptides prepared by bath ultrasound.•PV, RP, PL, D combined with hydrophobic amino acids were the structural characteristics of peptides.•Hydrogen bonding and cation-pi interaction were the main interaction modes between ACE and peptides. To reveal the structural characteristics and angiotensin-converting enzyme (ACE) inhibition mechanism of Stropharia rugosoannulata mushroom peptides prepared by multifrequency ultrasound, the peptide distribution, amino acid sequence composition characteristics, formation pathway, and ACE inhibition mechanism of S. rugosoannulata mushroom peptides were studied. It was found that the peptides in S. rugosoannulata mushroom samples treated by multifrequency ultrasound (probe ultrasound and bath ultrasound mode) were mainly octapeptides, nonapeptides, and decapeptides. Hydrophobic amino acids were the primary amino acids in the peptides prepared by ultrasound, and the amino acid dissociation of the peptide bonds at the C-terminal under the action of ultrasound was performed mainly to produce hydrophobic amino acids. Pro and Val (PV), Arg and Pro (RP), Pro and Leu (PL), and Asp (D) combined with hydrophobic amino acids were the characteristic amino acid sequence basis of the active peptides of the S. rugosoannulata mushroom. The docking results of active peptides and ACE showed that hydrogen bond interaction remained the primary mode of interaction between ACE and peptides prepared by ultrasound. The peptides can bind to the amino acid residues in the ACE active pocket, zinc ions, or key amino acids in the domain, and this results in inhibition of ACE activity. Cation–pi interactions also played an important role in the binding of mushroom peptides to ACE. This study explains the structural characteristics and ACE inhibition mechanism used by S. rugosoannulata mushroom peptides prepared by ultrasound, and it will provide a reference for the development and application of S. rugosoannulata mushroom peptides.