The tuberculosis (TB) global health crisis is aggravated by the rise of multi- and extensively-drug resistant Mycobacterium tuberculosis (Mtb). Shorter and more effective treatments as well as new TB drugs are urgent priorities for reducing the global burden of TB. Current TB treatment requires combinations of antibiotics, new TB treatment regimens are likely to comprise multiple agents, and new antibiotics are likely to be introduced to augment established combinations. Thus, new antitubercular candidates need to be assessed for potential beneficial and detrimental interactions with currently used TB antibiotics. Here, we examined interactions between TB antibiotics and wollamide B1, a chemically optimised derivative of wollamides, a new class of Streptomyces-derived cyclic hexapeptides with promising anti-mycobacterial activity and low mammalian cytotoxicity. Wollamide B1 exhibited anti-mycobacterial activity against phylogenetically diverse Mtb clinical and reference strains, including multi- and extensive-drug resistant clinical isolates. Fractional Inhibitory Combination Index and Zero Interaction Potency models of two- and multi-way interactions revealed that wollamide B1 synergises with first- and second-line TB antibiotics in inhibiting the replication and promoting the killing of Mtb. This was accompanied by Mtb transcriptional signatures unique to wollamide B1 and synergistic combinations, as revealed by RNA sequencing. Notably, wollamide B1 enhanced anti-mycobacterial activity of the combination of bedaquiline/pretomanid/linezolid, a recently introduced new regimen for multi-drug resistant TB. Collectively, our data deliver new insights into the anti-mycobacterial activity of wollamides that might inspire the development of new antibiotics for improved TB treatments.