Sulfur nanoparticles (S-NPs) were prepared in this work by precipitation method using sodium thiosulphate and tetraoctylammonium bromide surfactants in conc. hydrochloric acid media. The sizes and shapes of S-NPs were confirmed by scanning electron microscope (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD) techniques. Broth micro-dilution method was applied to determine antibacterial activity of S-NPs against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa reference strains. S-NPs exhibited antimicrobial activity (MIC = 5.47µg/ml) against Staphylococcus aureus strain (Gram-positive bacterium). On the other hand, no antimicrobial activity was detected against Gram-negative bacteria isolates (Escherichia coli and Pseudomonas aeruginosa) at 0.68 to 800 µg/ml.