Background Staphylococcus lugdunensis is a commensal skin microorganism that, unlike other coagulase-negative staphylococci, presents increasing clinical importance. This species yields a metalloprotease called lugdulysin that may contribute to its higher degree of virulence. This study aimed to determine the biochemical characterization of the lugdulysin produced by S. lugdunensis clinical isolates and investigate its effect on the formation and disruption of biofilm of Staphylococcus aureus isolates. The protease was isolated and characterized for its optimal pH and temperature, activity in the presence of inhibitors and enzymatic kinetics. The influence of metal cofactor supplementation on proteolysis was also evaluated, with and without inhibitors. Finally, the protease capacity to inhibit and disrupt biofilms of different S. aureus lineages and biofilm matrix was analyzed. Results The protease optimal pH and temperature were 7.0 and 37° C, respectively. EDTA inhibited the protease, and the activity was not recovered by divalent ion supplementation. In addition, divalent ions did not change enzymatic activity without inhibitors, which was stable for up to 3 hours. Its structure was determined via homology modelling. The protease significantly inhibited the formation and disrupted established biofilms of S. aureus isolates with protein biofilm. Conclusions This study confirmed features of the lugdulysin metalloprotease and showed that this S. lugdunensis virulence factor may be a new competition mechanism and/or modulation of the staphylococcal biofilm.