This study aimed to evaluate the antimicrobial potential of the supernatant (conditioned medium) of mesenchymal stem cells-derived conditioned medium (MSCs CM), incorporated in chitosan nanoparticles (CS NPs), against multidrug-resistant (MDR) clinical isolates. Dynamic light scattering (DLS), zeta potential (ZP), and scanning electron microscopy (SEM) assays were used to characterize CS NPs. MSCs CM was incorporated into CS NPs, and its release pattern from the composite (MSCs CM-CS NPs) was evaluated. Antibacterial properties of MSCs CM-CS NPs, as well as MSCs CM and CS NPs, were assessed by the microbroth dilution technique. The average particle size of most CS NPs was 85.2 nm, and their zeta potential was 32.1 mV. SEM findings supported the morphology of the prepared CS NPs and the synthesized composite (MSCs CM-CS NPs). Entrapment efficiency of the new construct was 71%. The antimicrobial activity of the new construct (MSCs CM-CS NPs: 1000 μg/mL + 0.05%) against MDR bacteria was significantly higher compared with MSCs CM (1000 μg/mL) and CS NPs (0.05%) each alone. This composite exhibited the synergistic antibacterial activity of the combination of MSCs CM and CS NPs, resulting in a more potent dose-dependent antibacterial activity against MDR clinical isolates.