Aim: Purpose of the research was to assess the amount of microbial accumulation and further disinfection of mouthguards usually used in sports. Methodology: Decontaminating effect of diverse substances on EVA mouthguards hitherto contaminated with saliva and broth culture of Enterococcus faecalis and Candida albicans. Afterwards, the mouthguards were undertaken to the following treatments solutions (A) Untreated; (B) 5 min with sterilized distilled water (C) 5 min with H2O2 (D) 5 min with a physiological solution (E) toothbrush and fluoride toothpaste (F) 5 min with 0.5% NaOCl. Results: The maximum efficacy against E. faecalis was demonstrated by H2O2 (84.19% bacterial load reduction). H2O2 showed a better reduction of salivary cell load. The maximum efficacy against C. albicans was seen when immersed in 0.5% NaOCl which caused a 92.95% decrease of cell load. Conclusion: Hydrogen peroxide, 0.5% sodium hypochlorite allowed to obtain an optimum disinfection of the mouthguard.