The use of metal nanoparticles (NPs) to enhance hydrogen production in dark fermentation (DF) has become a pioneering field of interest. In particular, iron-based nanoparticles (FeNPs) play a pivotal role in enhancing the activity of metalloenzymes and optimizing feedstock utilization, resulting in improved hydrogen production. This study investigated the effect of FeNPs (magnetite) supplementation at three different concentrations of 50, 100, and 200 ppm in a continuous dark fermenter for the production of hydrogen from rice straw acid hydrolysate. The highest hydrogen production rate of 2.6 ± 0.3 NL H₂/L-d was achieved with the addition of 100 ppm of nanoparticles, representing a 53% increase compared to the condition without FeNPs addition. This improvement was driven by a microbial community in which Clostridium was the major dominant genus. In addition, increasing the nanoparticle concentration to 100 ppm resulted in an increase in butyrate concentration to 2.0 ± 0.1 g/L, which is 43% higher than the butyrate concentration without FeNPs. However, when the NP concentration was increased to 200 ppm, the hydrogen production rate decreased to 1.6 ± 0.2 NL H₂/L-d. This study can serve as a guideline for future research aimed at evaluating the effects of FeNPs in continuous dark fermentation systems. This work highlights the potential benefits and challenges associated with the use of FeNPs, paving the way for future studies to optimize their application and improve the efficiency of dark fermentation processes.