Phytochemical investigation of the methanolic extract of Bursera simaruba bark led to the isolation of 11 compounds, including lignans yatein, beta-peltatin-O-beta-D-glucopyranoside, hinokinin and bursehernin, and three natural compounds namely 3,4-dimetoxyphenyl-1-O-beta-D-(6-sulpho)-glucopyranoside, 3,4,5-trimetoxyphenyl 1-O-beta-D-(6-sulpho)-glucopyranoside and 3,4-diidroxyphenylethanol-1-O-beta-D-(6-sulpho)-glucopyranoside. Their structures were established by NMR and ESI/MS experiments. Additionally, an LC-ESI/MS qualitative study on the phenolic compounds and an LC-ESI/MS/MS quantitative study on the lignans found in the methanolic extract of B. simaruba bark were performed to give value to the plant as source of these biological active compounds. Quantitative analyses results confirmed that compounds yatein, beta-peltatin-O-beta-D-glucopyranoside, hinokinin and bursehernin are major compounds in the bark and, in particular, beta-peltatin-O-beta-D-glucopyranoside appears to be the most abundant.