We investigated the in vitro activities of short lipopeptides against a large panel of clinical isolates of antibiotic resistant bacteria. In the animal model, LP16 (5 mg/kg) significantly decreased the burden of viable colony forming unit (CFU) of bacteria. MTT assay results revealed the high selectivity of lipopeptides toward microbial cells. Calcein dye leakage experiments and flow cytometric analysis suggests the membranolytic effect of lipopeptides, which was further confirmed by visualizing bacterial damage via electron microscopy tool (SEM & TEM). Moreover, stability in human blood plasma and no sign of resistance development against clinical isolates of Escherichia coli and Staphylococcus aureus were observed for lead lipopeptides. These results demonstrate the potential of short lipopeptides as a novel class of anti-infectives.