Stenotrophomonas maltophilia is an emerging drug-resistant pathogen. Here, we demonstrated that S. maltophilia flagellin could locally activate innate immunity thereby increase the resistance to respiratory tract infection (RTI) and improved bacterial clearance in the lungs of BALB/c mice. The test group consisted of BALB/c mice instilled with 5 μg of purified flagellin and challenged 4 h later with S. maltophilia. In this group, increased production of pro-inflammatory cytokines (IL-1β and TNF-α), myeloperoxidase activity, caspase-1 activity and nitric oxide (NO) was seen in lung homogenates in significant amounts (P < 0.05) as compared to control groups. On the contrary, low level of malondialdehyde was detected in the test group. Activation of alveolar macrophages in terms of bacterial engulfment and intracellular bacterial killing at an early stage and delayed production of anti-inflammatory cytokine (IL-10) was also detected. Concomitant with elevation of pro-inflammatory mediators, high number of leukocytes infiltration was detected in bronchoalveolar lavage of treated mice. The generated mucosal immune response was found to be protective against S. maltophilia as well as Staphylococcus aureus infection. In conclusion, this study emphasizes the nonspecific protection mediated by flagellin against homologous as well as heterologous bacterium that might be exploited therapeutically to prevent the development of RTI.