Green technology for bio-hydrogen production refers to the use of electrochemical systems and electroactive bacteria. Microbial electrolysis cells (MECs) are one of these systems. Among the electroactive bacteria were used in this study Escherichia coli NRRL B-3008 and Enterobacter aerogenes DSM 30053. Water from Al-Salam Canal was also used as a substrate according to chemical structure (H2O) of water as cheap source to produce hydrogen gas by using microorganisms i.e bacteria. The result obtained were indicate that Escherichia coli NRRL B-3008 gave the highest values of bio-hydrogen production rates (Bio-HPR = 102.7 cm3) and hydrogen yield (YH2 = 57.19 %) was obtained from MEC (anode 500ml) and an applied voltage 0.8V. Enterobacter aerogenes DSM 30053 gave the highest values of Bio-HPR = 132.57 cm3 and YH2 = 70.13 % was obtained from MEC (anode 500ml) and an applied voltage 0.4V. While, the highest volume of Bio-H2 without bacteria was 51.1 cm3 and YH2 = 31.18 % with an applied voltage 0.8 V (anode 500ml). Therefore, the bacterial electrolysis are very important biological process for highest hydrogen yield by bio-hydrogen production.