This work investigated hydrogen production from biomass feedstocks (i.e., glucose, starch, lignin and cellulose) using a 100 mL h-type proton exchange membrane electrolysis cell. Biomass electrolysis is a promising process for hydrogen production, although low in technology readiness level, but with a series of recognised advantages: (i) lower-temperature conditions (compared to thermochemical processes), (ii) minimal energy consumption and low-cost post-production, (iii) potential to synthesise high-volume H2 and (iv) smaller carbon footprint compared to thermochemical processes. A Lewis acid (FeCl3) was employed as a charge carrier and redox medium to aid in the depolymerisation/oxidation of biomass components. A comprehensive analysis was conducted, measuring the H2 and CO2 emission volume and performing electrochemical analysis (i.e., linear sweep voltammetry and chronoamperometry) to better understand the process. For the first time, the influence of temperature on current density and H2 evolution was studied at temperatures ranging from ambient temperature (i.e., 19 °C) to 80 °C. The highest H2 volume was 12.1 mL, which was produced by FeCl3-mediated electrolysis of glucose at ambient temperature, which was up to two times higher than starch, lignin and cellulose at 1.20 V. Of the substrates examined, glucose also showed a maximum power-to-H2-yield ratio of 30.99 kWh/kg. The results showed that hydrogen can be produced from biomass feedstock at ambient temperature when a Lewis acid (FeCl3) is employed and with a higher yield rate and a lower electricity consumption compared to water electrolysis.