We report the isolation and anti-allergic activity (degranulation reduction) of six polyphenolic compounds, one of them being novel, from olive mill wastes (OMW). Additionally, we evaluated their ability to reduce; intracellular Ca2+ levels and expression of calcium channel proteins in RBL-2H3 cells, to assist the possible characterization of the mechanisms involved. The novel compound, new hemialdehydic decarboxymethylated oleuropein aglycone, HDOA 1 (IC50 = 108.3 mu M), hydroxytyrosol acetate 3 (IC50 = 40.2 mu M) and 1-acetoxypinoresinol 4 (IC50 = 43.8 mu M) reduced intracellular Ca2+ levels after challenging by both A23187 and DNP-BSA, suggesting a mechanism beyond just blockage of extracellular Ca2+ influx. While luteolin 2, despite its strongest activity (IC50 = 3.9 mu M), reduced Ca2+ levels only after challenging by A23187, suggesting its main mechanism is by blocking extracellular Ca2+ influx. We confirmed further, that, each compound decreased the expression of, two or more, calcium channel proteins suggesting that they act as 'mast cell stabilizers' by reducing the expression of calcium channel proteins. This work attracts more attention to OMW as a potential source of lead compounds for structural modification to improve and develop new anti-allergic drugs.