Biological methanation is driven by anaerobic methanogenic archaea, cultivated in different media, which consist of multiple macro and micro nutrients. In addition, a reducing agent is needed to lower the oxidation–reduction potential (ORP) and enable the growth of oxygen-sensitive organisms. Until now, sodium sulfide (Na2S) has been used mainly for this purpose based on earlier published articles at the beginning of anaerobic microbiology research. In a continuation of earlier investigations, in this study, the usage of alternative reducing agents like sodium dithionite (Na2S2O4) and L-Cysteine-HCl shows that similar results can be obtained with fewer environmental and hazardous impacts. Therefore, a newly developed comparison method was used for the cultivation of Methanothermobacter marburgensis. The median methane evolution rate (MER) for the alternatives was similar compared to Na2S at different concentrations (0.5, 0.25 and 0.1 g/L). However, the use of 0.25 g/L Na2S2O4 or 0.1 g/L L-Cys-HCl led to stable MER values over consecutive batches compared to Na2S. It was also shown that a lower concentration of reducing agent leads to a higher MER. In conclusion, Na2S2O4 or L-Cys-HCl can be used as a non-corrosive and non-toxic reducing agent for ex situ biological methanation. Economically, Na2S2O4 is cheaper, which is particularly interesting for scale-up purposes.