Piperine (PPN) is a known inhibitor of efflux pumps in Mycobacterium tuberculosis and in vitro synergism with rifampicin (RIF) has been proven. The current study evaluates the activity of PPN and synergism with RIF in rapidly and slowly growing nontuberculous mycobacteria (NTM). Also, to propose a possible mechanism of interaction of PPN with M. leprae (Mlp) RNA polymerase (RNAp). Minimal inhibitory concentration and drug combination assay was determined by resazurin microtiter assay and resazurin drug combination assay, respectively. In silico evaluation of PPN binding was performed by molecular docking and molecular dynamics (MD). PPN showed higher antimicrobial activity against rapidly growing NTM (32-128 mg/L) rather than for slowly growing NTM (≥ 256 mg/L). Further, 77.8% of NTM tested exhibited FICI ≤ 0.5 when exposed to PPN and RIF combination, regardless of growth speed. Docking and MD simulations showed a possible PPN binding site at the interface between β and β’ subunits of RNAp, in close proximity to the trigger-helix and bridge-helix elements. MD results indicated that PPN binding hindered the mobility of these elements, which are essential for RNA transcription. We hypothesize that PPN binding might affect mycobacterial RNAp activity, and, possibly, RIF activity and that this mechanism is partially responsible for synergic behaviors with RIF reported in vitro. Communicated by Ramaswamy H. Sarma