Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) and still remains a major health problem worldwide. Recently, the need to develop new and more effective drugs to treat emerging multidrug-resistant TB (MDR-TB) and to reduce side effects of anti-TB drugs has been recognized, such as toxicity in liver and many other detrimental changes. In this study, to develop a novel candidate drug for the effective TB treatment with fewer side effects in host, the mycobactericidal activity of pasakbumin A (5F) was examined in H37Rv-infected macrophages. 5F is an extracted natural compound from Tongkat ali and it is known as healthy tonic, aphrodisiac property, anti-cancer and anti-malaria treatment. 5F had not direct toxicity to Mtb and macrophage cells, however, it significantly suppressed intracellular bacterial growth in Mtb-infected cells. 5F caused a significant increase in TNF- α production and a decrease in IL-10 production in Mtb-infected cells. The secretion of NO was also markedly enhanced in 5F -treated cells during Mtb infection. In addition, 5F treatment strongly induced the phosphorylation level of ERK1/2 and IκBα after 15 min of infection. Compared with Mtb-infected cells treated with 5F alone, combined treatment of 5F and anti-TB drug, rifampicin (RMP), was more effective in the inhibiting intracellular bacterial growth and IL-10 production and increasing TNF- α production. Furthermore, the ratio of conversion from LC3-I to LC3-II and endogenously expressing LC3, known as indicator of autophagy, rose in Mtb-infected cells treated with 5F and RMP. Treatment of 5F and RMP also potently induced the phosphorylated levels of ERK1/2, compared with Mtb-infected cells treated with 5F alone. These findings revealed that 5F could be developed as a novel TB drugs or adjuvants to improve mycobactericidal activity through the enhancing autophagy pathway and the increase of NO and inflammatory cytokine production.