Bovine mastitis, which in most cases has S. aureus and S. agalactiae as causal agents, is an important disease in the livestock sector of Brazil due to losses it originates in the milk production chain and derivatives. In particular, S. aureus has a high zoonotic potential and is often associated as a cause of food poisoning outbreaks. Thus, controlling such microorganism in cows suffering from mastitis becomes crucial. However, this is an arduous task, since this bacterium has features that end up protecting it during conventional therapies, often resulting in failed treatments. The use of polymeric nanocarriers in drug delivery system has the potential to increase the effectiveness of the treatment of this disease, mainly due to the orientation of those active drug carrying nanostructures to within the cells of the mononuclear phagocyte system. In this compartment, antibiotic concentrations needed to tackle the pathogen are hardly achieved. This project aimed to develop a nanostructured formulation for intramammary use containing cloxacillin for use in clinical trials phase II and III. The working team included researchers from Embrapa, UFJF (Federal University of Juiz de Fora), and members of FAPEMIG (Minas Gerais State Research and Innovation Support Foundation)-sponsored Nanobiotechnology Network, which were represented by UFOP (Federal University of Ouro Preto) and UFMG (Federal University of Minas Gerais). The results open a perspective, as a multi-thematic research line, to the production of more efficient nanostructured antibiotic formulations for intramammary use, thereby minimizing treatment failures and the emergence of bacterial resistance.