Phytochemical investigation of the stem bark of Stereospermum acuminatissimum K. Schum. resulted in the isolation of 21 compounds, including two new guanine derivatives, 1,3,7-trimethylguanin-1/3-ium (1) and 3,7-dimethylguanin-1/3-ium (2), and one new phenolic long chain ester, 2-(4-hydroxyphenyl)ethyl hentriacontanoate (3). The known compounds were identified as sterequinones A, F, and H (4, 5, and 6), zenkequinones A-B (7-8), p-coumaric acid (9), methyl caffeate (10), caffeic acid (11), psilalic acid (12), syringaldehyde (13), norviburtinal (14), specioside (15), verminoside (16), tyrosol (17), eutigoside A (18), ellagic acid (19), atranorin (20), and ursolic acid (21). The metabolites were screened for their potential against urease and α-chymotrypsin enzymes, as urease is targeted in peptic ulcer while α-chymotrypsin is used to remove protein debris in ulcer. Compound 20 was found to be excellent urease inhibitor with IC(50) value of 18.2 ± 0.03 μM. Compounds 13 and 18-20 are reported for the first time from the genus Stereospermum. The chemotaxonomic significance of the isolated compounds was also described.