The effect of the Phenoloxidase (PO) on bacterial culture and its bioactivity is a big concerns nowadays because of the movement towards natural antibiotics instead of the chemical ones. In this study the prophenoloxidase (PPO) was activated to PO in Spodoptera littoralis by injection of series concentrations from Bacillus thuringiensis kurstaki (BT)  (3200 IU/mg, AGERIN- wettable powder) 2X10¹⁰, 2X10²⁰, 2X10³⁰, 2X10⁴⁰ , 2X10⁵⁰ and 2X 10⁶⁰ cells/ml. 10 µl of each bacterial concentration was injected into groups from 10 larvae each, with a total 656 larvae. 519 of the injected larvae were live and 137 died. It was found that the stock concentration 2X10⁶⁰ was the LC20 concentration. After the injection and inoculation for 24 hrs, the larvae had been disinfected and the haemolymph separated.  Purification for PO from the haemolymph had been done using HiTrapTM CM FF 1ml column. After assuring the presence of PO by SDS gel, it was cultured against six types of bacteria, two gram +ve (Staph. aureus, Enterococci) and four gram –ve (E. coli, Pseudomonas, Acinetobacter and Klebsiella). It was effective against the gram positive bacteria and against E. coli only from the gram negative bacteria. Therefore, it could be concluded that the PO has bioactivity toward the gram positive bacteria more than the gram negative ones.