Toward the design of new proline-rich peptidomimetics, a short peptide segment, present in several proline-rich antimicrobial peptides (AMPs), was selected. Fatty acids of varying lengths and spermine were conjugated at the N- and C-terminals of the peptide, respectively. Spermine-conjugated lipopeptides, C<sub>10</sub>-PR-Spn and C<sub>12</sub>-PR-Spn, exhibited minimum inhibitory concentrations within 1.5-6.2 μM against the tested pathogens including resistant bacteria and insignificant hemolytic activity against human red blood cells up to 100 μM concentrations and demonstrated resistance against trypsin digestion. C<sub>10</sub>-PR-Spn and C<sub>12</sub>-PR-Spn showed synergistic antimicrobial activity against multidrug-resistant methicillin-resistant <i>Staphylococcus aureus</i> with several tested antibiotics. These lipopeptides did not permeabilize bacterial membrane-mimetic lipid vesicles or damage the <i>Escherichia coli</i> membrane like the nonmembrane-lytic AMP, buforin-II. The results suggested that C<sub>10</sub>-PR-Spn and C<sub>12</sub>-PR-Spn could interact with the 70S ribosome of <i>E. coli</i> and inhibit its protein synthesis. C<sub>10</sub>-PR-Spn and C<sub>12</sub>-PR-Spn demonstrated superior clearance of bacteria from the spleen, liver, and kidneys of mice, infected with <i>S. aureus</i> ATCC 25923 compared to levofloxacin.