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Edible flowers have gained attention as unconventional food sources due to their nutritional and functional properties. This study provides novel information on the chemical composition, cytotoxicity and antiproliferative effects of Theobroma mariae flowers. The objective of this paper was to identify bioactive compounds in its flowers using one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). The phenolic fraction of the flowers revealed bioactive compounds such as hyperoside, guaijaverin, astragalin, juglalin, and kaempferol. The results confirmed the potential of T. mariae flowers as a source of phenolic compounds, emphasizing their feasibility for possible applications in the development of functional foods. Moreover, the antiproliferative assay demonstrated that the phenolic fraction inhibits cell proliferation (GI50) while presenting low cytotoxicity in both cancer and normal cells.