NZ17074 (N1), an arenicin-3 derivative isolated from the lugworm, has potent antibacterial activity and is cytotoxic. To reduce its cytotoxicity, seven N1 analogues with different structures were designed by changing their disulfide bonds, hydrophobicity, or charge. The "rocket" analogue-N2 and the "kite" analogue-N6 have potent activity and showed lower cytotoxicity in RAW264.7 cells than N1. The NMR spectra revealed that N1, N2, and N6 adopt β-sheet structures stabilized by one or two disulfide bonds. N2 and N6 permeabilized the outer/inner membranes of E. coli, but did not permeabilize the inner membranes of S. enteritidis. N2 and N6 induced E. coli and S. enteritidis cell cycle arrest in the I-phase and R-phase, respectively. In E. coli and in S. enteritidis, 18.7-43.8% of DNA/RNA/cell wall synthesis and 5.7-61.8% of DNA/RNA/protein synthesis were inhibited by the two peptides, respectively. Collapsed and filamentous E. coli cells and intact morphologies of S. enteritidis cells were observed after treatment with the two peptides. Body weight doses from 2.5-7.5 mg/kg of N2 and N6 enhanced the survival rate of peritonitis- and endotoxemia-induced mice; reduced the serum IL-6, IL-1β and TNF-α levels; and protected mice from lipopolysaccharide-induced lung injury. These data indicate that N2 and N6, through multiple selective actions, may be promising dual-function candidates as novel antimicrobial and anti-endotoxin peptides.