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Bitter melon extract has been used as a traditional medicine for several ailments. Modern techniques have authenticated its use for diabetes and also as an antibacterial, antiviral, anthelmintic and abortifacient agent. In this study, cytotoxicity of bitter melon seed powder extracts from water, ethanol, and a water- ethanol (1:1) mixture were tested on HEK293 cells and red blood cells (RBC). The water-ethanol extract showed the highest cytotoxic activity in inhibiting cell growth, but had less cytotoxic activity on RBC cell lysis. Whereas the ethanol extract had less cytotoxic activity in inhibiting cell growth but lysed RBC completely. To identify the active components in the water-ethanol extract, several separation methods were performed, and cytotoxicity was tested on HEK293 cells and RBC. The first method used ammonium sulfate precipitation and both the supernatant and precipitate were collected. The second method used size exclusion filtration and the filtrates were collected. Two fractions were isolated with highest cytotoxicity activity on cells. The fourth method used high performance liquid chromatography (HPLC), and several fractions were collected. The various fractions were tested on HEK293 cells and RBC. Two HPLC fractions exhibited their highest toxicity on HEK293 cells. Gas chromatography-mass spectroscopy (GC-MS) analysis of these two HPLC fractions identified potentially new type of low molecular weight cytotoxic compounds.