The two-peptide (class IIb) bacteriocins are generally thermostable small-molecule (< 10 kDa) two-component antimicrobial peptides produced by Gram-positive bacteria. This class of peptides mainly rely on peptide-peptide interactions mediated by typical motifs to form active dimeric transmembrane proteins. Numerous studies have shown that two-peptide bacteriocins have reliable safety and desirable bacteriostatic effect, holding great potential in the control of drug-resistant bacteria. Therefore, the structural features and action mechanisms of two-peptide bacteriocins have received considerable research attention. From the perspectives of the structure formation of two-peptide bacteriocins, peptide-peptide interaction, and peptide-membrane interaction, this article summarizes the mechanism of action of this class of antimicrobial peptides. Meanwhile, the structural regularity of two-peptide bacteriocins and the structural features affecting their activities are elaborated by synthesizing current research. This review will provide new ideas for future research on two-peptide bacteriocins.