Objective: To evaluate whether the inoculation of contaminated cultures in the peritoneal cavity of mice could implement decontamination of Acanthamoeba cultures. Methods: Suspensions of Acanthamoeba, Acanthamoeba polyphaga ATCC 30461, or Acanthamoeba spp. isolated from soil (UnB13 strain) were inoculated in the peritoneal cavity of Swiss mice (n=24). After 1, 6, 12, or 24h of exposure the peritoneal cavity was washed and assessed for the presence of bacteria, fungi, and Acanthamoeba. Results: After 1h of intraperitoneal inoculation at least 97% of the bacteria and 96% of the fungi (P<0.05) and 99% of the bacteria (P<0.05) were successfully eliminated from the ATCC 30461 strain and from the soil isolate UnB13 strain, respectively. This method also allowed the recovery of most trophozoites and cysts from both Acanthamoeba cultures at the end of 24h. Conclusions: Our data demonstrated that this technique has great potential for decontamination of Acanthamoeba cultures in a short period of time.