Facing the continuously urgent demands for novel antimicrobial agents since the growing emergence of bacterial resistance, a series of new ultra-short lipopeptides, composed of tryptophan and arginine and fatty acids, were de novo designed and synthesized in this study. Most of the new lipopeptides exhibited preferable antimicrobial potential against gram-positive bacteria, including MRSA clinical isolates. Among them, the new lipopeptides C14-R1 (C14-RWW-NH) and C12-R2 (C12-RRW-NH) presented higher selectivity to bacterial membranes over mammalian membranes and low cytotoxicity, which also maintained better antimicrobial activity in the presence of physiological salts or serum. Most importantly, C14-R1 and C12-R2 not only expressed low tendency of bacterial resistance, but also displayed synergistic antimicrobial activity against antibiotics-resistant bacteria when be used in combination with antibiotics. Especially, they could alleviate or reverse the ciprofloxacin resistance, implying an ideal anti-resistance function. Moreover, the new lipopeptides showed rapid killing kinetics, obvious effectiveness for persistent cells that escaped from antibiotics, and strong anti-biofilm ability, which further indicated a preferable anti-resistance ability. The typical non-receptor-mediated membrane mechanisms were characterized by LPS/LTA competitive inhibition, cytoplasmic membrane depolarization, PI uptake assay and scanning electron microscopy analyses systematically. Reactive oxygen species (ROS) generation assays supplemented their intracellular targets in the meanwhile. In addition to the remarkable antimicrobial activity in vivo, the new lipopeptides also displayed significant anti-inflammatory effect in vivo. To sum up, the new lipopeptides C14-R1 and C12-R2 viewed as novel antimicrobial alternatives for tackling the impending crisis of antimicrobial resistance.