Lipopeptides are medicinally essential building blocks with strong hemolytic, antifungal and antibiotic potential. In the present research article, we are presenting our findings regarding the synthesis of N–alkylated lipopeptides via Ugi four-component approach, their antimicrobial potential against pathogenic (Gram-positive and Gram-negative) bacteria, as well as computational studies to investigate the compounds binding affinity and dynamic behavior with MurD antibacterial target. Molecular docking demonstrated the compounds have good binding ability with MurD enzyme. The FT94, FT95 and FT97 compounds revealed binding affinity scores of −8.585 kcal mol− 1, −7.660 kcal mol− 1 and −7.351 kcal mol− 1, respectively. Furthermore, dynamics analysis pointed the systems high structure dynamics. The docking and simulation results were validated by binding free energies, demonstrating solid intermolecular interactions and in the assay in vitro, the Minimal Inhibitory Concentration (MIC) of FT97 to Staphylococcus aureus (S. aureus) was 62.5 μg/mL. In conclusion, a moderate inhibitory response of peptoid FT97 was observed against the Gram-positive bacteria, S. aureus and B. cereus.