Copper nanoparticles (Cu-NPs) were synthesized using Murraya koenigii leaf extract as reducing agent and characterized. TEM images show that the Cu-NPs were mostly cuboid shaped with an average size of about 97 nm. The influence of phytogenic Cu-NPs on biohydrogen production from glucose was studied using Enterobacter cloacae and Clostridium acetobutylicum, and its effects were compared with CuSO4. The effects of Cu-NPs and CuSO4 (2.5, 5.0, 7.5, 10.0 and 12.5 mg/L) on biohydrogen production using E. cloacae at pH 7.0 and C. acetobutylicum at pH 6.0 were performed in batch mode. Both Cu-NPs and CuSO4 supplementation inhibited the biohydrogen production in a dose dependent manner. The Cu-NPs were found to have more inhibitory effect on biohydrogen production of E. cloacae and C. acetobutylicum than CuSO4. (C) 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.