Sulfur is an abundantly available biologically active element and has been used for a long time as a bactericidal and fungicidal drug in both medical, veterinary and in agricultural practices. Synthesized hydrophilic sulfur nanoparticles can potentially allow the delivery of high concentrations of sulfur to infections. In the present study, a quick and environmentally friendly approach has been developed for the synthesis of hydrophilic sulfur nanoparticles with antibacterial activity. hydrophilic sulfur nanoparticles (SNPs) were prepared by modifying the surface of elemental sulfur by various water-soluble surfactants including hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl benzene sulfonate (SDBS) and Triton X-100 (TX-100). The hydrophilic SNPs were characterized by UV–visible spectrophotometer, X-ray diffraction (XRD), and thermogravimetric analysis (TG/DSC), scanning electron microscope (SEM), the hydrophilic SNPs showed an almost spherical shape with an average size in the range of 150-200 nm. The antibacterial activity of the hydrophilic sulfur nanoparticles was tested using against Gram-positive (S. aureus and E. faecium) and Gram-negative (Escherichia coli and P. aeruginosa) bacteria. As a result, that hydrophilic SNPs exhibited significant antimicrobial activity against Gram-positive bacteria, i.e., S. aureus and E. faecium. The acute toxicity of the hydrophilic SNPs was studied at a single oral administration of solutions of hydrophilic SNPs, as a result of the study of hydrophilic SNPs, it was determined as a non-toxic substance. The received products can be considered as perspective forms of application in agriculture and medicine.