A new stigmasterone 7αj 1 5α-dihydroxystigmast-4-en-3-one (1), along with fifteen known compounds was isolated from the EtOAc and n-BuOH fractions of the EtOH extract of Agathis macrophylla. The structure of these compounds 1-16 were established mainly by spectroscopic analysis and by comparison of the spectroscopic data with those reported for structurally related compounds. The isolated compounds 1-8 were screened for cytotoxicity in A549 tumor cell line using RSB assay. Compounds 1 and 2 displayed moderate cytotoxicity against A549 tumor cell line with IC₅₀ value of 36.5 and 16.0 μmol/L, respectively.