In Vitro: ML-SA1 (25 μM; 0~14 hours; A549 cells) possibly affects the entry of DENV2 into host cells. ML-SA1 (0~200 μM; A549 cells) shows that there is no cytotoxicity to the cell line observed, even at concentrations up to 200 μM. ML-SA1 (0~50 μM; A549 cells) significantly suppresses DENV2 at the RNA levels and the IC50 is 8.93 μM. ML-SA1 results in a dose-dependent decrease in ZIKV in A549 cells at both the RNA and protein levels, and the IC50 value of ML-SA1 against ZIKV RNA is 52.99 μM. ML-SA1, as an activator of TRPMLs, appears to be a potent inhibitor of DENV2 and ZIKV in vitro. ML-SA1 promotes lysosomal acidification and protease activity to inhibit viral infection. ML-SA1 can induce autophagy in Huh7 cells or A549 cells.