The secreted Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (MptpB) is an essential virulence factor required for the intracellular survival of Mtb within host macrophages. MptpB has become a promising target for the development of novel anti-tuberculosis (TB) drugs. In this study, two new fusarielins, fusarielins M (1) and N (2), and a biogenetically related known compound, fusarielin G (3) were isolated from the marine-derived fungus Fusarium graminearum SYSU-MS5127. Their inhibitory effects on MptpB were evaluated. Among these compounds, fusarielin M substantially inhibited MptpB with a half-maximal inhibitory concentration (IC) of 1.05 ± 0.08 μM, and an inhibition constant (K) of 1.03 ± 0.39 μM. Surface plasmon resonance analysis was used to characterize the interaction between fusarielin M and MptpB in vitro. Fusarielin M also exhibited cellular activity in blocking MptpB-mediated Erk1/2 and p38 inactivation in macrophages. Importantly, fusarielin M (20 μM) substantially reduced intracellular mycobacterial growth within macrophages, causing a 62% reduction in the bacterial burden. The binding mode of fusarielin M was further explored via molecular docking which suggested that fusarielin M binds to the active site of MptpB, forming a hydrogen bond with the side chain of Asp165; this is unique in the P-loop of MptpB compared to conventional human PTPs. The contact between fusarielin M and Asp165 in the catalytic loop provides a potential basis for inhibitor selectivity. Therefore, fusarielin M shows great potential as an anti-TB drug candidate.