Microbiota studies have dramatically increased over these last two decades, and the repertoire of microorganisms with potential health benefits has been considerably enlarged. The development of next generation probiotics from new bacterial candidates is a long-term strategy that may be more efficient and rapid with discriminative in vitro tests. Streptococcus strains have received attention regarding their antimicrobial potential against pathogens of the upper and, more recently, the lower respiratory tracts. Pathogenic bacterial strains, such as non-typable Haemophilus influenzae (NTHi), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus), are commonly associated with acute and chronic respiratory diseases, and it could be interesting to fight against pathogens with probiotics. In this study, we show that a Streptococcus mitis (S. mitis) EM-371 strain, isolated from the buccal cavity of a human newborn and previously selected for promising anti-inflammatory effects, displayed in vitro antimicrobial activity against NTHi, P. aeruginosa or S. aureus. However, the anti-pathogenic in vitro activity was not sufficient to predict an efficient protective effect in a preclinical model. Two weeks of treatment with S. mitis EM-371 did not protect against, and even exacerbated, NTHi lung infection.