Heterocapsa triquetra (Ht), a non-toxic dinophyte, was selected to investigate the presence of pigments inhibiting melanoma cells growth. We developed an innovative microwave-assisted extraction process (MAE), determined the Ht pigment composition using Reverse Phase-High Performance Liquid Chromatography (HPLC) and high-resolution Ultra Performance Liquid Chromatography-Mass Spectrometry E (UPLC-MSE) analysis and assessed their antiproliferative activity in human invasive melanoma cells (A2058) grown in vitro. MAE allowed a fast and efficient extraction of Ht pigments as compared with conventional processes (soaking, sonication). Seventeen pigments and derivatives were unequivocally identified by UPLC-MSᴱ in the Ht extract, among which two were supposed to be produced during the extraction (hydroxylated chlorophyll a and pheophytin a). Pigment composition was fully coherent with that usually described for the DINO-1 peridiniales order. It included the uncommon carotenoids P457, peridininol, peridinin, pyrrhoxanthin, diadinochrome and diadinoxanthin. Ht pigment ethanol extract induced 18% growth inhibition of A2058 malignant melanoma cell line after a 72 h treatment at 100 μg·mL− 1 and pigment purification confirmed the highly significant antiproliferative activity of chlorophyll c2, peridinin, dinoxanthin and diatoxanthin on this cell line. Our data demonstrate for the first time the effectiveness of microwave irradiation to extract pigments from dinophytes. They also indicate that Heterocapsa triquetra and other dinophytes are interesting sources to purify bioactive pigments with antiproliferative activities in chemoresistant melanoma cells. These results strongly support previous findings about the anticancer activity of phytoplankton pigments and reinforce the possible interest of these molecules as natural cytostatic drugs.