Four Gram-negative bacteria (E.coli ATCC 19404, P. aeruginosa ATCC9027, A. hydrophila ATCC 7966 and V. damsel ATCC 33539) were screened for production of endotoxin. P. aeruginosa recoded the highest endotoxin concentration (338.59 x 10³ IU/ml). The optimization of endotoxin production resulted in the increase of endotoxin titer (25% at pH 7.0 and 26% at both 30°C and 90 rpm). The detection of lipid core complex (50 kDa) and O-specific polysccahrides (20, 25, 38 kDa) was performed by the application of SDS-PAGE. GC/MS analysis indicated the presence of 4 compounds namely 2H Pyaran, 2,5 diethenyltetrahydro, Tetradecanoic acid, Bis(2 ethylhexyl) phthalate and Dodecanoic acid. The endotoxin was thermally stable (78%) at 120°C for 60 min. Reduction of endotoxin activity (45 and 69%) occurred when treated with 1N-NaOH or HCl for 60 min, respectively. The endotoxin showed a bio-toxicity against Artemia salina, (LC₅₀of 102.99mg/ml). The endotoxin exhibited antitumor activity against colon cancer cells (Caco-2) and breast cancer cells (Mcf-7) with growth reduction amounted to87% and 92%, respectively. On the other hand, a weak antiviral activity (5.2%) was recorded in Vero cells infected with HAV. In conclusion, the endotoxin with antioxidant property could be an alternative for the treatment of colon and breast cancer.