Objective: The goal is to biosynthesize sulphur nanoparticles (Sulphur NPs)from Bacillus coagulans extract and test its bioactivity against S. aureus gram positive bacteri that was isolated from dermatitis patients. Methods: 125 specimens were taken from patients of dermatitis attended two Baghdad hospitals. Among 125 specimens, only 32 of isolates were identified as S. aureus. Five S. aureus isolates biofilm forming(strong) and multi drug resistant(MDR) were selected. Fourteen types of antibiotics from different classes were used and the results showed different patterns of resistance. Congo red agar (CRA) and microtiter plate method (MPT) were used to estimate the ability of MDR isolates of S. aureus to create biofilms. A series of characterization techniques, including UV–vis, FTIR, FE-SEM, EDS, XRD and TEM analysis, have been carried out to characterize the biosynthesized sulfur NPs. The antimicrobial, antibiofilm and antioxidant activities of sulfur NPs were estimated. Results: Sulfur NPs biosynthesized exhibited antimicrobial activity against examined bacteria with inhibition zone ranged between 12 to 20, 18 to 23 and 22 to 26mm at 100, 200 and 300µg/ml, respectively. Only five strong-biofilm producers and MDR of S. aureus(A5, A8, A9, A11 and A29) were used to assess the ability of sulfur NPs as anti-biofilm agent. No significant differences (p>0.05) in inhibition rate of all strain isolates (A5, A8, A9, A11 and A29) for all concentrations of Sulfur NPs with p=0.886. Conclusions: The antioxidant activity of sulfur NPs were decreased into 81.6, 80.3, 75.2 and 68.9% with using concentrations of 400, 300, 200 and 100ug/ml, respectively, also, nanoparticles had the potential to scavenge DPPH free radicals.