Mycobacterium tuberculosis (Mtb) is the aetiological agent of tuberculosis, the leading cause of death worldwide from a single infectious agent. Mtb is a highly adaptable human pathogen that might enter a dormant non-replicating (NR), drug-tolerant stage. Reactivation of dormant Mtb can lead to active disease. Antibiotic treatments of active and latent tuberculosis are long, complex and may fail to fully eradicate the infection. Therefore, it is imperative to identify novel compounds with new mechanisms of action active against NR bacilli. Dormant Mtb habitat is mostly thought to be the pH-neutral and hypoxic caseous granuloma. We have used the Wayne culture model to reproduce this environment and tested the activities of two DNA-targeted agents, C8-linked-pyrrolobenzodiazepine(PBD)-polyamide conjugates 1 and 2, against Mtb grown in aerobic and hypoxic conditions in both acidic and pH-neutral media. PBD 2 showed growth inhibitory activity at 5.1 µg/ml against 19-day-old hypoxic NR Mtb cultures with 1.8 log10 CFU reduction on day 21 at pH 7.3. PBD 2 was particularly effective against 5-day-old aerobic cells at pH 7.3, with CFU reduction (>6.8 log10) on day 21 at 5.1 µg/ml being identical to that of rifampin at 8 µg/ml. PBD 2 qualifies as a promising lead against aerobic and NR Mtb.