Encapsulation is a promising technology to carry natural active substances, preventing their loss and maintaining their stability until use. Beads of chitosan-containing propolis have been prepared using a mono-pore filter device, which permits the encapsulation of natural polyphenols avoiding heat treatments, high shear rates and the use of toxic solvents. Beads proved to be active against Bacillis cereus, Escherichia coli, Listeria innocua, Pseudomonas fluorescens, Yarrovia lipolytica and three moulds strains; the highest effect was found against Staphylococcus aureus (MIC 0.8 mg beads mL−1). Results in liquid cultures of S. aureus evidenced that beads were able to release the flavonoids from propolis: the diffusion of the active compounds is a key factor in the exploitation of the microbial activity. The obtained chitosan–propolis beads represent an example of natural antimicrobial delivery system that could be used to prevent the growth of pathogenic/spoilage bacteria in food applications.