Cryptosporidium is a ubiquitous harsh protozoan parasite that resists many disinfectants. It remains viable and infective for a long time in water and food causing global outbreaks. Chitosan (the deacetylated chitin molecule) was used in its nanosuspension form to evaluate its effect against Cryptosporidium parvum. The experiments were performed in vitro in serial concentrations and confirmed in mice in vivo infectivity assay. Chitosan nanoparticles (Cs NPs) were toxic to Cryptosporidium oocysts. The effect appeared to decrease the number of Cryptosporidium oocysts and altered their content. The destruction rate of oocysts was dependent on the dose of chitosan and the time of exposure (P < 0.05). Higher doses of Cs NPs over a prolonged period exhibited a significantly higher destruction rate. Using staining and light microscopy, remarkable destructive changes were observed in the oocysts' morphology. The minimal lethal dose for > 90% of oocysts was 3000 μg/ml, no mice infections in vivo were observed. The results in this study elucidate Cs NPs as an effective anti-cryptosporidial agent.