The peptide α-helix is right-handed when containing amino acids with l-chirality, and left-handed with d-chirality, however mixed chirality peptides generally do not form α-helices unless a helix inducer such as the non-natural residue amino-isobutyric acid is used. Herein we report the first X-ray crystal structures of mixed chirality α-helices in short peptides comprising only natural residues as the example of a stapled bicyclic and a linear membrane disruptive amphiphilic antimicrobial peptide (AMP) containing seven l-and four d-residues, as complexes of fucosylated analogs with the bacterial lectin LecB. The mixed chirality α-helices are superimposable onto the homochiral α-helices and form under similar conditions as shown by CD spectra and MD simulations but non-hemolytic and resistant to proteolysis. The observation of a mixed chirality α-helix with only natural residues in the protein environment of LecB suggests a vast unexplored territory of α-helical mixed chirality sequences and their possible use for optimizing bioactive α-helical peptides.