In recent years the application of metal nanoparticles is gaining attention in various fields. The present study focuses on the additive effect of `green’ synthesized iron nanoparticles (FeNPs) on dark fermentative hydrogen (H2) production by a mesophilic soil bacterium Enterobacter cloacae. The FeNPs were synthesized by a rapid green method from FeSO4 using aqueous leaf extract of Syzygium cumini. The synthesized FeNPs showed a characteristic surface plasmon resonance peak at 267 nm. The transmission electron microscopy images confirm that the formation of FeNPs was mainly porous and irregular in shape, with an average particle size of 20–25 nm. The presence of iron (Fe) in the synthesized FeNPs was confirmed by energy-dispersive X-ray spectroscopy. The comparative effect of FeSO4 and FeNPs on batch fermentative H2 production from glucose was investigated. The fermentation experiments reveal that the percentage and yield of H2 in FeNPs supplementation were increased significantly than the control (no supplementation) and FeSO4 containing media. The maximum H2 yield of 1.9 mol mol−1 glucose utilized was observed in 100 mg l−1 FeNPs supplementation, with two-fold increase in glucose conversion efficiency. Thus, the result suggests that FeNPs supplementation in place of FeSO4 could improve the bioactivity of H2 producing microbes for enhanced H2 yield and glucose consumption.