Treatment of Staphylococcus aureus infections remains very difficult due to its capacity to survive intracellularly and its multidrug resistance. In this study, the extracellular/intracellular activities of plectasin derivatives-MP1102/NZ2114 were investigated against three methicillin-susceptible/-resistant S. aureus (MSSA/MRSA) strains in RAW 264.7 macrophages and mice to overcome poor intracellular activity. Antibacterial activities decreased 4-16-fold under a mimic phagolysosomal environment. MP1102/NZ2114 were internalized into the cells via clathrin-mediated endocytosis and macropinocytosis and distributed in the cytoplasm; they regulated tumor necrosis factor-α, interleukin-1β and interleukin-10 levels. The extracellular maximal relative efficacy (Emax) values of MP1102/NZ2114 towards the three S. aureus strains were >5-log decrease in colony forming units (CFU). In the methicillin-resistant and virulent strains, MP1102/NZ2114 exhibited intracellular bacteriostatic efficacy with an Emax of 0.42-1.07-log CFU reduction. In the MSSA ATCC25923 mouse peritonitis model, 5 mg/kg MP1102/NZ2114 significantly reduced the bacterial load at 24 h, which was superior to vancomycin. In MRSA ATCC43300, their activity was similar to that of vancomycin. The high virulent CVCC546 strain displayed a relatively lower efficiency, with log CFU decreases of 2.88-2.91 (total), 3.41-3.50 (extracellular) and 2.11-2.51 (intracellular) compared with vancomycin (3.70). This suggests that MP1102/NZ2114 can be used as candidates for treating intracellular S. aureus.