The EtOAc-soluble fraction of the MeOH extract of propolis afforded a new prenylated chromane derivative, 3-hydroxy-2,2-dimethyl-8-prenylchromane-6-propenoic acid (1), along with 22 known compounds, 2−23. Of the known compounds, 4, 7, 12−19, and 22 were isolated for the first time from propolis, and the absolute configuration of 23 was established as (2S,3R). Investigation suggested that Baccharis spp. are a significant source of tropical Brazilian propolis, in addition to Clusia minor, Clusia major, and Araucaria heterophylla. All the compounds were tested for their cytotoxicity toward human HT-1080 fibrosarcoma and murine colon 26-L5 carcinoma cells. Among these compounds, 9 and 19−21 showed potent cytotoxicity, having ED₅₀ values equal to or less than 10 μg/mL.