In this paper, nanosuspensions of three arsenic sulfide (As4S4) compounds, commercial synthetic arsenic(II) sulfide, and natural realgar and pararealgar minerals were prepared. Nanosuspensions were obtained by ultrafine wet milling in a circulation mill. The zeta potential and particle size distribution were measured for stability estimation. Structural changes were studied using Raman and Fourier transform infrared spectroscopic methods and positron annihilation lifetime method. The morphology of the prepared nanoparticles was determined using scanning and transmission electron microscopy. The anticancer effects were tested using flow cytometry and western blotting analysis. The average particle size in the individual samples varied from 137 to 153 nm. The effects of milling were associated with the formation of arsenic sulfide crystalline nanoparticles and the fragmentation of the corresponding free-volume entities. Consequently, an increase in the arsenic dissolution was observed. The anti-cancer effects of the nanosuspensions were verified on the human cancer H460 cell line, in which case DNA damage and greater numbers of apoptotic cells were observed.