Probiotics might provide an alternative approach for the control of oral candidiasis. However, studies on the antifungal activity of probiotics in the oral cavity are based on the consumption of yogurt or other dietary products, and it is necessary to use appropriate biomaterials and specific strains to obtain probiotic formulations targeted for local oral administration. In this study, we impregnated gellan gum, a natural biopolymer used as a food additive, with a probiotic and investigated its antifungal activity against 28.4, a strain recently isolated from the oral cavity of a caries-free individual, was incorporated in several concentrations of gellan gum (0.6% to 1% [wt/vol]). All tested concentrations could incorporate cells while maintaining bacterial viability. Probiotic-gellan gum formulations were stable for 7 days when stored at room temperature or 4°C. Long-term storage of bacterium-impregnated gellan gum was achieved when 28.4 was lyophilized. The probiotic-gellan gum formulations provided a release of cells over 24 h that was sufficient to inhibit the growth of , with effects dependent on the cell concentrations incorporated into gellan gum. The probiotic-gellan gum formulations also had inhibitory activity against sp. biofilms by reducing the number of sp. cells ( < 0.0001), decreasing the total biomass ( = 0.0003), and impairing hyphae formation ( = 0.0002), compared to the control group which received no treatment. Interestingly, a probiotic formulation of 1% (wt/vol) gellan gum provided an oral colonization of in mice with approximately 6 log CFU/ml after 10 days. This formulation inhibited growth ( < 0.0001), prevented the development of candidiasis lesions ( = 0.0013), and suppressed inflammation ( = 0.0006) compared to the mice not treated in the microscopic analysis of the tongue dorsum. These results indicate that gellan gum is a promising biomaterial and can be used as a carrier system to promote oral colonization for probiotics that prevent oral candidiasis.