Mouthguards in Ethylene Vinyl Acetate (EVA) should be sanitized to prevent alteration of oral microbiota. The present study determined, in vitro and by SEM observation, the decontaminating effect of different substances on EVA mouthguards previously contaminated with saliva and broth culture of Enterococcus faecalis ATCC 29212 and Candida albicans CH 34 (clinically isolated). Subsequently, the mouthguards were subjected to the following treatments: (A) Untreated; (B) 5 min with sterilized distilled water (HO d); (C) 5 min with HO; (D) 5 min with a physiological solution; (E) toothbrush and fluoride toothpaste; (F) 5 min with 0.5% NaOCl; (G) 5 min with Oral Care Foam™; (H) 5 min with Bite Sept™. The highest efficacy against E. faecalis was demonstrated by HO (84.19% bacterial load reduction). HO and Oral Care Foam™ showed a greater reduction of salivary cell load. The highest efficacy against C. albicans was demonstrated by 0.5% NaOCl which caused a 92.95% reduction of cell load. In conclusion, hydrogen peroxide, 0.5% sodium hypochlorite and the solution Oral Care Foam™ allowed to obtain an optimum disinfection of the mouthguard. SEM observation showed that different substances demonstrated a decontaminating effect decreasing the microbial communities on the EVA surface.