The present study assessed the efficiency of utilizing mixed culture bacteria (MCB) incorporated with individual nanoparticles (NPs), i.e., hematite (α-Fe2O3), nickel oxide (NiO), and zinc oxide (ZnO), dual NPs (α-Fe2O3 + NiO, α-Fe2O3 + ZnO, and NiO + ZnO), and multi-NPs (α-Fe2O3 + NiO + ZnO) for hydrogen production (HP) from industrial wastewater containing mono-ethylene glycol (MEG). When MCB was individually supplemented with α-Fe2O3 (200 mg/L), NiO (20 mg/L), and ZnO NPs (10 mg/L), HP improved significantly by 41, 30, and 29%, respectively. Further, key enzymes associated with MEG metabolism, such as alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and hydrogenase (hyd), were rapidly and substantially enhanced in the medium. NiO and ZnO NPs notably promoted ADH and ALDH activities, respectively, while α-Fe2O3 exhibited superior impact on hyd activity. Maximum hydrogen production rate was concomitant with higher acetic acid production and lower residual acetaldehyde and ethanol. HP using MCB supplemented with individual NiO (20 mg/L) and ZnO NPs (10 mg/L) further improved by 8.0%-14% when dual and multi-NPs were used; the highest HP was recorded when multi-NPs were used. In addition, NPs incorporation resulted in substantial increase in the relative abundance of Clostridiales (belonging to family Clostridiaceae; > 83%). Overall, this study provides significant insights into the impact of NPs on hydrogen production from MEG-contaminated wastewater.