Shorter and more effective treatments as well as new drugs with novel molecular targets and mechanisms of action are urgent priorities for reducing the global burden of drug-sensitive and drug-resistant tuberculosis (TB). Treatment of TB requires combinations of antibiotics for extensive periods of time. Thus, new drugs are likely to be introduced as part of multi-drug regimens. Wollamides are a new class of Streptomyces-derived cyclic hexapeptides with anti-mycobacterial activity and we previously demonstrated wollamide synergism with select TB antibiotics, including delamanid. To begin to define the wollamide mechanism of action and the molecular basis for wollamide synergism with delamanid, we assessed the Mycobacterium tuberculosis (Mtb) transcriptional response to wollamides alone, or in combination with delamanid. Wollamide exposure rapidly induced expression of a gene cluster indicative of cell wall perturbations. Consistent with this transcriptional response, we observed compromised mycobacterial cell envelope integrity upon wollamide exposure. Prolonged exposure of Mtb to wollamides induced a transcriptional signature that was unique when compared to currently used TB antibiotics, which suggests a novel mechanism of action. Exposure of Mtb to the synergistic wollamide-delamanid combination revealed dominance of the wollamide-induced transcriptional response and also revealed additional signatures unique to the combination. Collectively, these data deliver new insights into the anti-mycobacterial activity of wollamides that might inspire the development of new antibiotics for improved TB treatments.