Daptomycin is an important antibiotic reserved to treat infections caused by multi-drug resistant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Daptomycin targets bacterial cell membranes for bactericidal effects. However, daptomycin resistance is emerging and associated with deep-seated infections. Novel therapeutic strategies are required to use daptomycin. Here, we utilized lipid-based nanoparticles, namely cubosomes, to encapsulate daptomycin for delivery and potentiate bactericidal effects via lipid-mediated interactions. Daptomycin-loaded cubosomes (Dp-Cub) released daptomycin at sustained rates up to 3 days, and exhibited synergistic killing of 14 clinical MRSA isolates in vitro. Dp-Cub significantly reduced bacterial burden of MRSA in organs in a murine septicemia model compared with daptomycin monotherapy, confirming this Dp-Cub synergistic effects in vivo. This Dp-Cub synergy was mediated by cubosomes docking on S. aureus cell surface, releasing daptomycin for membrane extraction and penetration, followed by infusion of cubosomes into S. aureus membranes. Together, these data illustrated that Dp-Cub are a promising system to combat Gram-positive bacterial infections.