Mycobacterium tuberculosis (M. tuberculosis) is causative pathogen of tuberculosis, and could induce long-term asymptomatic infection, known as latent tuberculosis infection (LTBI), in which the bacteria are thought to persist in a dormant state. It is reported that there are pairs of toxin-antitoxin systems (TASs) in chromosomes of M. tuberculosis. Some TASs play roles in the latent infection because they could not only regulate the bacterial growth, but also induce the bacteria to enter dormant state. Some TASs are suggested to be involved in the biofilm formation and stress reaction, but how they regulate biofilm formation has not yet been elucidated. M. tuberculosis in biofilm is resistant to many drugs and host immune protections. M. tuberculosis in dormant state is usually multidrug-resistant. These issues bring challenges for the treatment of tuberculosis. This review focuses on the research progress on TAS and biofilm formation of M. tuberculosis, as well as the effect of anti-tuberculosis drugs on biofilm formation.