The anaerobic digestion (AD) of biomass is a green technology with known environmental benefits for biogas generation. The biogas yield from existing substrates and the biodegradability of biomasses can be improved by conventional or novel enhancement techniques, such as the addition of iron-based nanoparticles (NPs). In this study, the effect of different concentrations of Fe2O3-based NPs on the AD of brown macroalga Sargassum spp. has been investigated by 30 days trials. The effect of NPs was evaluated at different concentrations. The control sample yielded a value of 80.25 ± 3.21 NmLCH4/gVS. When 5 mg/g substrate and 10 mg/g substrate of Fe2O3 NPs were added to the control sample, the yield increased by 24.07% and 26.97%, respectively. Instead, when 50 mg/g substrate of Fe2O3 NPs was added to the control sample, a negative effect was observed, and the biomethane yield decreased by 38.97%. Therefore, low concentrations of Fe2O3 NPs favor the AD process, whereas high concentrations have an inhibitory effect. Direct interspecies electron transfer (DIET) via Fe2O3 NPs and their insolubility play an important role in facilitating the methanogenesis process during AD.