Antibiotic resistant bacterial infection is currently a serious public concern. Their ability to form biofilms further complicates the treatment. Herein we investigated the activity of lipo-cyclic γ-AApeptides against both planktonic cells and biofilms of Staphylococcus epidermidis and Pseudomonas aeruginosa, in comparison to those of the conventional antibiotic ciprofloxacin. Our results suggest that these lipo-cyclic γ-AApeptides exhibit comparable or enhanced performance compared to ciprofloxacin in the prevention of biofilm formation for both Gram-positive and Gram-negative bacteria, providing a potential alternative treatment and prevention for indwelling device-related infections.