Sports mouthguards have the potential to become a microbial reservoir, to produce oral and systemic diseases and cause changes in environmental oral factors, inhibiting the protective effect of saliva. The aim of this study was to monitor, in vivo, oral environmental changes caused by chlorhexidine and sports mouthguards and in vitro, the effects of microbial strains, saliva and chlorhexidine on Ethylene-Vinyl-Acetate (EVA) material. Seventy-two athletes were analyzed at different time points: before training session (T0), post-training (TA), post-training with mouthguard (TB), post-training with mouthguard and chlorhexidine (TC). At each time of observation, saliva was collected and subjected to microbiological analysis. In vitro, EVA disks were incubated with bacterial cultures, saliva and clorexidine. Culture of supernatant solution, SEM and bacterial counts of EVA disks were performed. S. mutans and Candida spp. load decreased significantly in TC. The pH value significantly decreased in TB and improved in TC. In vitro, the analyzed bacteria were organized to form a biofilm on the EVA disk surface. The addition of chlorhexidine to the bacterial culture and saliva inhibited the growth in all tested conditions. In vivo, the use of chlorhexidine associated with the sports mouthguard inhibited the growth of pathogenic microbial species, and improved pH values. In vitro, EVA stimulated biofilm formation on its surface, but this action was contrasted by chlorhexidine. The effects found in vitro encouraged the use of chlorhexidine in vivo as a valuable tool in the use of mouthguards.