Homogenous chemical modification of ball-milled eucalyptus wood with palmitoyl chloride was investigated using a dimethyl sulfoxide/N-methylimidazole solvent system at room temperature. The parameters were optimized, including the reaction time and the dosage of palmitoyl chloride. The results indicated that prolonging the reaction time from 15 min to 120 min resulted in an increase in the weight percentage gain (WPG) of eucalyptus from 9.5 to 22.8%, while a further increase of the reaction time to 180 min led to an increase in WPG of 19.3%. An increase of the mass ratio of palmitoyl chloride-to-wood from 1:2 to 5:1 resulted in an improvement of WPG from 8.7 to 46.0%, while a further increase of the mass ratio of palmitoyl chloride-to-wood to 6:1 led to a decrease in WPG (39.7%). The physico-chemical properties of the esterified wood were investigated with FT-IR and CP/MAS 13C-NMR spectroscopies. The results indicated that the palmitoyl ester group was successfully attached to the eucalyptus. The esterification of lignin and carbohydrates occurred at room temperature and the thermal stability of eucalyptus increased after esterification.