Tuberculosis (TB) is an airborne disease that kills 1.5 million people and affects ~ 9 million worldwide. Its etiological agent, Mycobacterium tuberculosis (Mtb), persists in a third of the general population. There is also an emergent problem of drug and multidrug resistance in Mtb that creates the need for novel drugs. In the treatment against TB, one target of antibiotics is the prokaryotic RNA polymerase (RNAP), an essential multitask enzyme that catalyzes RNA synthesis during transcription. Rifampicin, the first-line antibiotic anti-TB, binds and inhibits the MtbRNAP.Consequently, combatting TB requires a better understanding of the primary mechanism of transcription by MtbRNAP. In fact, during Mtb transcription, unique and essential factors might affect the MtbRNAP dynamics and regulate its activity. That regulation plays a significant role in the adaptation and pathogenesis of the parasite. Therefore, understanding how these factors and new antibiotics modulate together the MtbRNAP activity will contribute valid information to address TB. To this end, we use a single-molecule approach based on high-resolution optical tweezers to study transcription elongation by MtbRNAP in the context of its DNA elements, transcriptional factors, and antibiotics.