This study describes the development of propolis nanoparticles (PNP) to treat bovine mastitis. Three PNP prepared with varying concentrations of propolis (5% and 7%, w/v) and the surfactants [poloxamer (1%, 3%, and 4%, w/v) and soy lecithin (0.25%, 0.7%, and 1%, w/v)]. PNP were characterized according to their size, polydispersity, zeta potential, pH, morphology, and physical stability. PNP were evaluated for their in vitro antimicrobial and cytotoxic effects. PNP obtained were spherical with a monodisperse distribution (polydispersity index < 0.2) and an average particle size between 181 and 201 nm. Stability studies showed that PNP were stable over 150 d. The encapsulation efficiency of total phenolic content varied between 73% and 91%. The chromatographic profile of phenolic compounds from PNP showed selective encapsulation efficiency according to the polarity of compounds. All PNP showed antimicrobial activity against Staphylococcus aureus with a minimum inhibitory concentration ranging from 156 to 310 μg mL⁻¹. The IC₅₀ (the concentration responsible for reduction of cellular viability by half) for epithelial cells of bovine mammary gland (MAC-T, mammary alveolar cell-T) varied from 122.2 to 268.4 μg mL⁻¹. Results showed that PNP represent a promising nanocarrier for high concentrations of propolis extract in a stable aqueous medium, while, at the same time, presenting antimicrobial activity accompanied by moderate cytotoxicity to the MAC-T cells.