Sulfur nanoparticles (S-NPs) were prepared using rosemary (Rosmarinus officinalis) leaves aqueous extract and sodium thiosulfate pentahydrate (Na2S2O3.5H2O) at room temperature. The S-NPs were characterized by UV–visible spectrophotometer, Fourier transform infrared (FT-IR), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), and X-ray diffraction (XRD) techniques. The sulfur nanoparticles are crystalline in nature with average size of 40 nm. The morphology of S-NPs could be controlled by tuning the amount of rosemary leaves aqueous extract and sulfur ions. The cytotoxic effects of the S-NPs on hatching of second stage root-knot and mortality of second stage of root-knot nematode (Meloidogyne javanica) were tested. These results confirmed that the S-NPs synthesized using rosemary leaves aqueous extract and could possibility be used as anti-nematocide to manage M. javanica infestation.