Nontuberculous mycobacteria are ubiquitous and saprophytic organisms that have been implicated in a wide spectrum of diseases due to an increasing number of immunocompromised patients. The natural resistance of atypical mycobacteria to classical antituberculous drugs has encouraged research into new chemotherapeutic agents and drug combinations. The aim of this study was to determine the in vitro antimycobacterial activities of β-lapachone alone and in combination with isoniazid against
Mycobacterium fortuitum
and
Mycobacterium smegmatis
via the Time-Kill Curve method. A 2 log
10 CFU/mL reduction in the
M. smegmatis culture was observed 72 h after adding β-lapachone at its minimum inhibitory concentration. This drug sterilised the culture in 120 h. For
M. fortuitum, a reduction of 1.55 log
10 CFU/mL occurred in 24 h, but regrowth was seen in contact with β-lapachone. Both microorganisms were resistant to isoniazid. Regrowth of
M. fortuitum and
M. smegmatis was observed at 48 h and 72 h, respectively. In combination, these two drugs had a bactericidal effect and sterilised both cultures in 96 h. These results are valuable because antibiotic-resistant bacteria are a major public health problem.