Abstract The prevalence of pulmonary nontuberculous mycobacterial (NTM) infections caused by Mycobacterium abscessus complex (MABSC) species is increasing worldwide and poses a particular threat to susceptible individuals with structural or functional lung conditions such as cystic fibrosis (CF), chronic obstructive pulmonary disease and bronchiectasis. The intrinsic recalcitrance of these pathogens to chemotherapeutic treatments, and alarming treatment failure rates place a high priority on the development of more effective treatment approaches. Although MABSC is known to form microaggregates or biofilms in the thickened alveolar walls, airways and lung cavity of patients, the precise contribution of biofilm formation to MABSC infection and recalcitrance to drug treatment has never been clearly established. Using clinically approved drugs thought to target a key transcriptional regulator (DosRS) required for mycobacteria to enter a state of non-replicating persistence and which we recently found to be potent inhibitors of MABSC biofilm formation, this application proposes to determine, for the first time, whether adding a biofilm inhibitor to standard antibiotic regimens may improve cure. Proof-of-concept of the therapeutic benefit of this approach with clinically-used drugs could be a short route to the clinic. Aim 1 will first test the hypothesis that, through its involvement in inducing a state of persistence in response to hypoxia, the two-component regulator DosRS contributes to the ability of MABSC to form biofilms and develop phenotypic drug tolerance in a chronic CF (b-ENaC-Tg) mouse model of MABSC infection recently developed at Colorado State University. Aim 2 will validate DosRS as the bona fide target of the biofilm inhibitors in MABSC. Finally, Aim 3 will assess the adjunct therapeutic potential of these biofilm inhibitors in MABSC- infected b-ENaC-Tg mice.