Staphylococcus aureus is a leading cause of a wide range of clinical chronic infections mainly due to the establishment of a biofilm. Biofilm, a population of bacteria within a self-produced matrix of extracellular polymeric substance, decreases the susceptibility to antibiotics, immune defenses and contributes to antimicrobial resistance. To date antibiotic combination has been considered a strategy to combat S. aureus infection, but this approach does not solves the main pharmacokinetic problem caused by biofilms, consisting in insufficient drug penetration within the structure. Therefore, new antimicrobial agents that could overcome this resistance need to be discovered. Fighting staphylococcal resistance and biofilm formation is an important goal of the pharmaceutical research. Some fungicide has been observed to have antibacterial effect. anyway their use as antibiotics on S.aureus has been poorly studied. The aim of this work was to investigate the effect of the fungicide itraconazole (IT) on S. aureus biofilm formation and explore by SEM the morphological alteration after treatment. A strong biofilm disaggregation and morphologically different extracellular vesicles (EV) production were observed starting from sublethal IT doses. This suggests that IT resistance phenomena on the part of S. aureus are more difficult to establish respect other antibiotics. The adjuvant properties of IT could be used to combat bacterial biofilm and/or to improve antibiotic treatment. Moreover, because the production of EV represents a secretory pathway involved in intercellular communication shared to mammalian cells, fungi, and bacteria, our study is important to increase information that can be generalized to higher organisms. Published by Elsevier Ltd.