The intracellular pathogen R. equi causes pneumonia in foals and people that closely resembles that caused by Mycobacterium tuberculosis (Mtb). Development of resistance to antimicrobials by R. equi and Mtb represents a major health problem for veterinary and human medicine. There is a need for control methods and treatment alternative to the use of antibiotics. Our alternative approach is to use a TLR2/6 and TLR9 agonist combination known as PUL-042 to stimulate innate immunity in the lungs. Our objectives were to evaluate safety and ex vivo efficacy of PUL-042 (Pulmotect Inc, Houston, TX) in newborn foals. Ten newborn foals were divided in 2 groups (PUL-042 and saline control) and submitted to a bronchoalveolar lavage (BAL) on day 2 of age, 6 nebulizations in a 2-week interval and a second BAL 24 h after the last nebulization. BAL cells were infected with a virulent strain of R. equi, and then lysed at either 0 h (T0) and after 48 h of culture (T48). Supernatants were saved for determination of INF-, IL-1, and IL-10 concentration by ELISA. PUL-042 did not induce either adverse effects or airway inflammation in foals, as assessed by clinical examination and BAL cytology. At the tested doses, nebulization of PUL-042 did not significantly (P < 0.05) affect ex vivo killing capacity of R. equi by AMs of foals at 2 and 21 days of age relative to controls. Results of cytokines are pending. Repeat nebulization of newborn foals with PUL-042 did not induce any adverse effects nor an inflammatory response in the lungs. At the dose tested, PUL-042 did not affect the killing capacity of R. equi by AMs, but its effects on other immune cells, and at other doses, need further evaluation.