Combining antimicrobial peptides (AMPs) with cell-penetrating peptides (CPPs) has shown promise in boosting antimicrobial potency, especially against Gram-negative bacteria. We examined the CPP-AMP interaction with distinct bacterial types based on cell wall differences. Our investigation focused on AMPs incorporating penetratin CPP and dihybrid peptides containing both cell-penetrating TAT protein fragments from the human immunodeficiency virus and Antennapedia peptide (Antp). Assessment of the peptides TAT-AMP, AMP-Antp, and TAT-AMP-Antp revealed their potential against Gram-positive strains (, Methicillin-resistant (MRSA), and ). Peptides TAT-AMP and AMP-Antp using an amyloidogenic AMP from S1 ribosomal protein , at concentrations ranging from 3 to 12 μM, exhibited enhanced antimicrobial activity against . TAT-AMP and TAT-AMP-Antp, using an amyloidogenic AMP from the S1 ribosomal protein , at a concentration of 12 µM, demonstrated potent antimicrobial activity against and MRSA. Notably, the TAT-AMP, at a concentration of 12 µM, effectively inhibited () growth and displayed antimicrobial effects similar to gentamicin after 15 h of incubation. Peptide characteristics determined antimicrobial activity against diverse strains. The study highlights the intricate relationship between peptide properties and antimicrobial potential. Mechanisms of AMP action are closely tied to bacterial cell wall attributes. Peptides with the TAT fragment exhibited enhanced antimicrobial activity against , MRSA, and Peptides containing only the Antp fragment displayed lower activity. None of the investigated peptides demonstrated cytotoxic or cytostatic effects on either BT-474 cells or human skin fibroblasts. In conclusion, CPP-AMPs offer promise against various bacterial strains, offering insights for targeted antimicrobial development.