This study aims to investigate the effect of the manufacturing process cryomilling on the antibacterial effectiveness of a novel antibacterial agent ZnS. ZnS nanoparticles are getting attention for their potential antibacterial properties due to the fact that release of Zn ions has demonstrated promising preliminary effects when applied on skin wounds. Particle size is essential to achieve bacteria inhibition and elimination since it has been shown that antibacterial activity can be increased with reduced particle size, which results in higher surface to volume ratio. In this study, ZnS nanoparticles were synthesized using one-pot colloidal synthesis as well as using biological Sulfate Reducing Bacteria synthesis under anaerobic environment with proper media. Both types of ZnS were further processed through cryomilling after synthesis to reduce the particle size. Scanning electron microscopy and X-ray diffraction techniques were used to characterize the morphology and crystallinity of the ZnS nanoparticles. To assess bacteria inhibition and elimination, in vitro ZOI studies consisting of inoculating cellulose disks on agar plates with Staphylococcus aureus (S. aureus) followed by incubation for 24 hours were performed. In vitro biofilm models consisting of inoculating cellulose disks on well-developed S. aureus biofilm on agar plates followed by 24-hour incubation were also conducted. CLSM was employed to qualitatively observe the antibacterial effectiveness; statistical analysis was also performed to quantitatively study the effectiveness of ZnS as antibacterial agents by counting the residual CFU left on the cellular disks. Results showed that the ZnS nanoparticles possess very good antibacterial properties against S. aureus, and incorporating cryomilling enhances ZnS antibacterial effectiveness.