Isoprene is a key intermediate compound for the production of synthetic rubber and adhesives and is also used as a building block in the chemical industry. Traditionally, isoprene is obtained from crude oil during the refinery process. Nevertheless, plants and animals are also able to synthesize this important compound. This work compares two renewable approaches for isoprene production: by photosynthetic organisms (autotrophic microalgae/cyanobacteria) and by heterotrophic organisms (bacteria). These are two alternative pathways for the conventional isoprene production obtained from the petrochemical-based refinery process, which were assessed in this work using green metrics. Their performance was evaluated in terms of: material efficiency, energy efficiency, economic evaluation and land use. A 10-tonne scale was chosen to perform the green metrics evaluation for both biological processes leading to isoprene. For each process, a comparison was made between a scenario considering the highest isoprene produced reported in the literature and a scenario considering the maximum theoretical stoichiometric isoprene productivity.