There is a growing consumer demand for safe and functional foods with reduced chemical preservatives. In this scenario, the natural antimicrobials emerge as promising alternative to ensure food safety and prevent foodborne diseases. Lactoferrin and chitosan are GRAS biopolymers with antimicrobial properties and low toxicity. The aim of the present study was to produce lactoferrin (L) and chitosan (C) nanoparticles by ionic crosslinking with tripolyphosphate (TPP) in order to increase the antimicrobial activity of these compounds. The nanoparticles were synthesized by six different proportions of Lactoferrin (L), Chitosan (C) and TPP while the total biopolymer concentration was kept constant in 0,1% (w/v). All the samples were characterized by size, zeta potential, morphology, chemical interactions (FTIR) and antibacterial activity against S. aureus. The minimum inhibitory concentration (MIC) of 3.5L:5.5Q:1TPP and 4.5L:4.5Q:1TPP nanoparticles were lower (0,0370 and 0,0463 mg/ml, respectively) than that of pure chitosan (0,1053 mg/ml) and pure lactoferrin (2 mg/ml) which were related to it physicochemical properties such as higher zeta potentials values (+39.30 and +33.07 mV) and smaller sizes (81.87 and 97.67 nm) than other samples. The improved antimicrobial activity of nanoparticles is attributed to their increased surface area compared to its mass counterpart, which favored particle interactions with the negative charges of the bacterial surface. These results indicate that it is possible to expand the application of chitosan and lactoferrin as natural preservatives for foods through nano-aggregation.