Staphylococcus aureusis considered a high priority pathogen by the World Health Organization due to its high prevalence and the potential to form biofilms. Currently, the available treatments forS. aureusbiofilm-associated infections do not target the extracellular polymeric substances (EPS) matrix. This matrix is a physical barrier to bactericidal agents, contributing to the increase of antimicrobial tolerance. The present work proposes the development of lipid nanoparticles encapsulating caspofungin (CAS) as a matrix-disruptive nanosystem. The nanoparticles were functionalized with D-amino acids to target the matrix. In a multi-target nano-strategy againstS. aureusbiofilms, CAS-loaded nanoparticles were combined with a moxifloxacin-loaded nanosystem, as an adjuvant to promote the EPS matrix disruption. In vitro and in vivo studies showed biofilm reduction after combining the two nanosystems. Besides, the combinatory therapy showed no signs of bacterial dissemination into vital organs of mice, while dissemination was observed for the treatment with the free compounds. Additionally, the in vivo biodistribution of the two nanosystems revealed their potential to reach and accumulate in the biofilm region, after intraperitoneal administration. Thus, this nano-strategy based on the encapsulation of matrix-disruptive and antibacterial agents is a promising approach to fightS. aureusbiofilms.