The field of probiotic applications is rapidly expanding, including their use for the control of respiratory tract infections. Nevertheless, probiotics ability to colonize the lung environment and to compete with pulmonary pathogens is still a poorly investigated research area. In this study, we aimed to evaluate the adhesion ability of a number of commercial probiotic strains to the human lung epithelial cell line A549. Furthermore, we assessed probiotic ability to prevent host cell adhesion of one of the major lung pathogens in cystic fibrosis, Pseudomonas aeruginosa, and to reduce the pathogen-induced inflammatory response of human peripheral blood mononuclear cells (PBMCs) in terms of cytokine release. Lactobacillus acidophilus displayed the highest adhesion ability to A549 cells evaluated as percent of adhered bacteria compared to the inoculum. In agreement with such an observation, L. acidophilus was the most efficient in preventing adhesion to A549 cells of a P. aeruginosa isolate from CF sputum. Three-color fluorescence labeling of A549 cells, P. aeruginosa, and L. acidophilus, and confocal microcopy image analyses revealed a likely exclusion effect played by both live and UV-killed L. acidophilus towards P. aeruginosa. Such results were confirmed by CFU count. When co-cultured with PBMCs, both live and UV-killed L. acidophilus reduced the amount of IL-1β and IL-6 in culture supernatants in a statistically significant manner. Overall, the results obtained point to L. acidophilus as an interesting candidate for further studies for a potential aerogenous administration to control P. aeruginosa infections.