<p>Novel therapeutic agents for methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) need to be developed because of the growing antibacterial resistance against MRSA. This study aimed to design and develop novel cyclic heptapeptides as potential antibacterial agents with improved efficacy and safety profiles against MRSA. Here, we designed 100 novel cyclic heptapeptides, and it was examined computationally and validated through experimental methods. The best three cyclic heptapeptides, such as Tyr⁶, Gly⁶, and Ala⁶, showed the binding energy between − 10.9 and − 11.23&#xa0;kcal/mol. These three cyclic heptapeptides were synthesised with 94% purity using the solid-phase peptide synthesis (SPPS) strategy, showing the 18-mm zone of inhibition against MRSA in the study of biological activities. Antibacterial evaluation against MRSA, methicillin-sensitive <i>Staphylococcus aureus</i> (MSSA), and vancomycin-resistant <i>Staphylococcus aureus</i> (VRSA) revealed that cyclic heptapeptide Ala⁶ shown&#xa0;the strongest activity (minimum inhibitory concentration (MIC), 22.1–39.5&#xa0;µg/mL), comparable to moxifloxacin, followed by cyclic heptapeptide Gly⁶ (moderate activity) and Tyr⁶ (least activity). Time-kill assays showed that cyclic heptapeptide Ala⁶ achieved up to 89.9% colony-forming unit (CFU) reduction in MSSA at 4 × MIC within 5&#xa0;h. Hemolysis testing confirmed cyclic heptapeptide Ala⁶’s excellent biocompatibility (&lt; 3.23% at 100&#xa0;µg/mL), in contrast to cyclic heptapeptide Gly⁶ and Tyr⁶ (&gt; 9.3% at 10&#xa0;µg/mL), while moxifloxacin caused minimal hemolysis. The best peptide was further investigated through molecular dynamics (MD) simulations and density functional theory (DFT) studies.</p>