This study aimed to isolate the novel bacterial strains from intertidal sludge that could produce hydrogen and bioflocculant simultaneously. A bacterium named as strain XF-56 was isolated for high production of hydrogen and bioflocculant. The isolated strain was designated as Bacillus sp. XF-56 by 16S rRNA gene sequence analysis. Strain XF-56 could grow using various carbon sources and glucose was found to be favorable for production of hydrogen and bioflocculant. Moreover, this strain XF-56 was able to produce hydrogen and bioflocculant at initial pH from 5.0 to 10.0, with an optimum initial pH for hydrogen production at 7.0 and bioflocculant activity at 8.0 respectively. In addition, strain XF-56 showed the strong salt tolerance and could produce hydrogen and bioflocculant at the salt concentration from 0.4% to 6.0%. The hydrogen production and bioflocculant activity by strain XF-56 were 1.47 +/- 0.05 mol H-2/mol glucose and 93.5% in marine culture conditions, which increased to 1.79 +/- 0.06 mol H-2/mol glucose and 98.6% respectively in fresh ones. This result demonstrated that the hydrogen-producing bacterium XF-56 with simultaneous production of bioflocculant is beneficial for cost-effective bio-treatment process and has a wide application potential in actual fresh and marine wastewater. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.