This study was to investigate defense mechanisms on cassava induced by salicylic acid formulation (SA) against anthracnose disease. Our results indicated that the SA could reduce anthracnose severity in cassava plants up to 33.3% under the greenhouse condition. The β-1,3-glucanase and chitinase enzyme activities were significantly increased at 24 hours after inocula- tion (HAI) and decrease at 48 HAI after Colletotrichum gloeosporioides challenge inoculation, respectively, for cassava treated with SA formulation. Synchrotron radi- ation–based Fourier-transform infrared microspectros- copy spectra revealed changes of the C=H stretching vibration (3,000-2,800 cm−1), pectin (1,740-1,700 cm−1), amide I protein (1,700-1,600 cm−1), amide II protein (1,600-1,500 cm−1), lignin (1,515 cm−1) as well as mainly C–O–C of polysaccharides (1,300-1,100 cm−1) in the leaf epidermal and mesophyll tissues treated with SA formulations, compared to those treated with fungicide carbendazim and distilled water after the challenged inoculation with C. gloeosporioides. The results indicate that biochemical changes in cassava leaf treated with SA played an important role in the enhancement of structural and chemical defense mechanisms leading to reduced anthracnose severity.