The study of antimicrobial peptides (AMPs), which are considered a source of new antibiotics, have recently attracted more and more attention. AMPs combine several large groups of peptides with different spectra and different mechanisms of action on bacterial cells. Peptides that form alpha-helices are better studied; the mechanisms of antimicrobial action of peptides that form beta structures have been studied to a lesser extent. A further increase in AMPs bactericidal effect would be useful for the practical use of AMPs. One strategy used to increase the bactericidal effect is the dimerization of AMP, which is usually carried out via the chemical conjugation of monomers. In this work, the strategy of sequential monomer dimerization into a single polypeptide chain was applied with the example of beta-structured AMP polyphemusin I. Polyphemusin I is one of the most effective AMPs and, accordingly, a candidate for practical use as an antibiotic. Several possible mechanisms of its action have been described. The most developed of these are the formation of pores in the cell membrane and translocation through the membrane without pronounced damage. These mechanisms result in different predictions of the effect of polyphemusin I dimerization on its bactericidal properties. In this article, the possible structures of the monomer and dimer of polyphemusin I in the lipid membrane were analyzed with molecular modeling methods. A monomer and a dimer of polyphemusin I, in which two monomers are connected in series through a flexible linker into a single polypeptide chain, were chemically synthesized. Quantitative assessment of the antimicrobial action of the monomer and dimer on different bacterial cultures was carried out; no differences were observed. Combination of the results of molecular modeling and laboratory studies show that pore formation is not a likely mechanism of the action of polyphemusin I.