The ultrashort linear antimicrobial tetrapeptide BRBR-NH2 with an unnatural residue biphenylalanine (B) has potent and rapid antimethicillin-resistant Staphylococcus aureus (MRSA) activity but lacks hemolytic activity. The anti-MRSA activity of BRBR-NH2 is 8-fold more potent than that of WRWR-NH2 and 16-fold more potent than that of FRFR-NH2. However, how to influence their antimicrobial activities and mechanisms through the substitution of different aromatic hydrophobic residues is still unclear. In this work, to study the effects of varying hydrophobic interactions and membrane selectivities of BRBR-NH2, we performed multiple long-time (1000 ns) molecular dynamics (MD) simulations to investigate the interactions of a red blood cell (RBC) membrane and a Gram-positive bacterial cell membrane with three different tetrapeptides (BRBR-NH2, WRWR-NH2, and FRFR-NH2) under different ratios of peptides and lipids and also explored the changes in the membrane and structural characteristics of peptides. The binding energy results show that BRBR-NH2 interacts weakly with the RBC membrane, while not all BRBR-NH2 can be adsorbed to the RBC membrane surface. The MD simulation results produced significant local membrane thinning of multiBRBR-NH2 peptides in the Gram-positive bacterial cell membrane. An in-depth analysis of structural features and peptide-membrane interactions suggests that the aggregation of BRBR-NH2 on the membrane surface plays a crucial role in the destruction of the cell membrane. Taken together with the observed local membrane thinning, the in-depth analysis demonstrated that the interactions between the lipid bilayer and the BRBR-NH2 aggregation surface result in a local disturbance of the membrane structure. It can be concluded that the high anti-MRSA activity of BRBR-NH2 is attributed to the aggregation of BRBR-NH2 on the membrane surface. On the other hand, WRWR-NH2 and FRFR-NH2 peptides tend to bind with the membrane surface in a monomeric form and cover the membrane surface in a carpet-like manner. Therefor; these results provide an advanced microscopic understanding of how hydrophobic interactions or hydrophobic residues affect the antimicrobial activity and mechanism of antimicrobial peptides (AMPs).