Infections of the ears, paranasal sinuses, nose and throat are very common and represent a serious issue for the healthcare system. Bacterial biofilms have been linked to upper respiratory tract infections and antibiotic resistance, raising serious concerns regarding the therapeutic management of such infections. In this context, novel strategies able to fight biofilms may be therapeutically beneficial and offer a valid alternative to conventional antimicrobials. Biofilms consist of mixed microbial communities, which interact with other species in the surroundings and communicate through signaling molecules. These interactions may result in antagonistic effects, which can be exploited in the fight against infections in a sort of “bacteria therapy”. Streptococcus salivarius and Streptococcus oralis are α-hemolytic streptococci isolated from the human pharynx of healthy individuals. Several studies on otitis-prone children demonstrated that their intranasal administration is safe and well tolerated and is able to reduce the risk of acute otitis media. The aim of this research is to assess S. salivarius 24SMB and S. oralis 89a for the ability to interfere with biofilm of typical upper respiratory tract pathogens. To investigate if soluble substances secreted by the two streptococci could inhibit biofilm development of the selected pathogenic strains, co-cultures were performed with the use of transwell inserts. Mixed-species biofilms were also produced, in order to evaluate if the inhibition of biofilm formation might require direct contact. Biofilm production was investigated by means of a spectrophotometric assay and by confocal laser scanning microscopy. We observed that S. salivarius 24SMB and S. oralis 89a are able to inhibit the biofilm formation capacity of selected pathogens and even to disperse their pre-formed biofilms. Diffusible molecules secreted by the two streptococci and lowered pH of the medium revealed to be implied in the mechanisms of anti-biofilm activity. S. salivarius 24SMB and S. oralis 89a possess desirable characteristics as probiotic for the treatment and prevention of infections of the upper airways. However, the nature of the inhibition appear to be multifactorial and additional studies are required to get further insights.