The detailed exergy analysis of photobiological hydrogen production from syngas through water–gas shift (WGS) reaction via anaerobic bacteria Rhodospirillum rubrum was presented. Several exergetic parameters for the fermentation process were computed based on both the conventional exergy and eco‐exergy concepts. The microorganisms were supported using acetate as carbon source at various concentrations (0.5–3 g/L) in a CO‐enriched media in a batch culture and produced hydrogen by utilizing the harmful carbon monoxide. The results manifested that the cumulative rational exergetic efficiency based on the conventional exergy was lower than that obtained using the eco‐exergy concept. However, the cumulative exergy destruction based on the conventional exergy concept was higher than of the eco‐exergy theory. Therefore, it seems that the eco‐exergy concept was much more suitable as decision‐making tool to assess the sustainability of renewable energy projects containing living microorganisms. The highest cumulative process exergetic efficiency values were achieved at the acetate concentration of 1 g/L via the exergy and eco‐exergy concepts, at 0.5156% and 0.5139%, respectively. This revealed the suitability of this dosage for large‐scale biohydrogen production. Principally, the findings of this study can provide a theoretical framework for decision making on sustainability and renewability of the biological hydrogen production. © 2016 American Institute of Chemical Engineers Environ Prog, 35: 1166–1176, 2016