AbstractRifampicin (RFP) is a first‐line drug used to treat a variety of infections, including wound infections but has limitations in its use due to its toxicity. Hence, an urgent need exists for the development of suitable carriers for the delivery of the antibiotic. In this study, a novel approach is introduced for drug administration, employing stimulus‐responsive carriers to achieve an on‐demand strategy. This innovative method aims to minimize the dosage and frequency of drug administration, consequently lowering cytotoxicity levels. We used the lipases‐sensitive polycaprolactone (PCL) to produce nanocomposites loaded with rifampicin (PCL−RFP NPs). Nanoparticles were prepared by a single‐step emulsion solvent evaporation method. The size distribution of blank nanoparticles (PCL NPs) and PCL−RFP NPs were 172±30 nm and 229±58 nm, respectively. The liberation of RFP from PCL−RFP NPs was monitored over a period of 72 h in the absence and the presence of lipase was 9.46±0.24 % and 53.3±3.33 %, respectively, indicating responsive behavior. The minimum inhibitory concentration to lipase‐expressing Staphylococcus aureus (S. aureus) of PCL−RFP NPs was significantly improved compared to the free drug. Cytotoxicity tests using human dermal fibroblasts showed that the nanocomposites had better biocompatible when compared to the free drug. These findings indicate that the developed nanocomposite carriers have the potential to be promising candidates for delivering antibiotics in the field of biomedicine.