Bone infection is a devastating condition resulting from implant or orthopaedic surgery. Therapeutic strategies are extremely complicated and may result in serious side effects or disabilities. The development of enhanced 3D scaffolds, able to promote efficient bone regeneration, combined with targeted antibiotic release to prevent bacterial colonization, is a promising tool for the successful repair of bone defects. Herein, polymeric electrospun scaffolds composed of polycaprolactone (PCL) nanofibres decorated with poly(lactic-co-glycolic acid) (PLGA) particles loaded with rifampicin were fabricated to achieve local and sustained drug release for more efficient prevention and treatment of infection. The release profile showed an initial burst of rifampicin in the first six hours, enabling complete elimination of bacteria. Sustained and long-term release was observed until the end of the experiments (28 days), facilitating a prolonged effect on the inhibition of bacterial growth, which is in agreement with the common knowledge concerning the acidic degradation of the microparticles. In addition, bactericidal effects against gram negative (Escherichia coli) and gram positive (Staphylococcus aureus) bacteria were demonstrated at concentrations of released rifampicin up to 58 ppm after 24 h, with greater efficacy against S. aureus (13 ppm vs 58 ppm for E. coli). Cell morphology and cytocompatibility studies highlighted the suitability of the fabricated scaffolds to support cell growth, as well as their promising clinical application for bone regeneration combined with prevention or treatment of bacterial infection.