Giardia lamblia (G. lamblia) is the most widely known protozoan parasite that causes human gastrointestinal infection worldwide. Some natural compounds exhibited pivotal effects against different infectious diseases. In this research, the antigiardial activity and cytotoxicity of fungal chitosan, nano-chitosan, Rhamnus cathartica (R. cathartica) and emodin were evaluated in Balb/c mice. Genotyping of G. lamblia was assessed by PCR-RFLP technique. Different concentrations of mentioned compounds were used to check their antigiardial and cytotoxicity effects on human intestinal epithelial cells (HT-29) after 24, 48 and 72 h. The G. lamblia strain used in the current work was genotyped and revealed as an AII assemblage. All the concentration showed acceptable activity against G. lamblia cysts and trophozoites in comparison to the negative and positive controls (furazolidone and metronidazole) in vitro (P < 0.05). Giardia lamblia cysts were susceptible after treatment in all experiments in vivo in comparison to negative control (P < 0.05). Approximately, in most of the concentration, nano-chitosan and emodin were more effective than chitosan and R. cathartica, respectively (P < 0.05). The effects of exposure times in antigiardial and cytotoxicity effects were not statistically significant (P > 0.05). The maximum mortality rate (100%) was achieved at 100 and 50 µg kg−1 concentrations after 48 and 72 h of exposure time, respectively. Our results provide significant information about the new antigiardial agent and proposed the nano-chitosan and emodin for the development of new drugs against G. lamblia in the future.