There is increasing demand to develop antimicrobial peptides (AMPs) as next generation antibiotic agents, as they have the potential to circumvent emerging drug resistance against conventional antibiotic treatments. Non-natural antimicrobial peptidomimetics are an ideal example of this, as they have significant potency and in vivo stability. Here we report for the first time the design of lipidated γ-AApeptides as antimicrobial agents. These lipo-γ-AApeptides show potent broad-spectrum activities against fungi and a series of Gram-positive and Gram-negative bacteria, including clinically relevant pathogens that are resistant to most antibiotics. We have analyzed their structure-function relationship and antimicrobial mechanisms using membrane depolarization and fluorescent microscopy assays. Introduction of unsaturated lipid chain significantly decreases hemolytic activity and thereby increases the selectivity. Furthermore, a representative lipo-γ-AApeptide did not induce drug resistance in S. aureus, even after 17 rounds of passaging. These results suggest that the lipo-γ-AApeptides have bactericidal mechanisms analogous to those of AMPs and have strong potential as a new class of novel antibiotic therapeutics.