Nowadays, biodiesel is recognized as one of the best alternative to fossil fuels due to its renewability, environmentally-friendly nature and simple preparation from different feedstocks such as microalgae. In this work, biodiesel was produced from Chlorella vulgaris microalgae and its environmental performance was evaluated applying an exergo-environmental analysis. This process was simulated using Computer Aided Process Engineering (CAPE) tools in a commercial software to determine the main operating conditions of all streams. An exergy balance around the system was carried out to determine both exergy losses and exergy inlets, which were used to determine exergo-environmental parameters. The Sustainability Index (SI), Renewability Indicator (RI), Net Energy Ratio (NER), and Net Energy Value (NEV) were calculated. It was obtained a SI value of 8.18 indicating that the input exergy is higher than the exergy destruction. The NEV and NER parameters were 38,846.31 MJ/kg BD and 1.19, respectively. The effect of use of fossil fuel-derived energy on the RI was also analyzed and it was found an improvement opportunity in this parameter by increasing the percentage of non-renewable energy substitution. These results revealed that third-generation biodiesel production from 2202 María Ochoa-García et al. microalgae represents an exergy efficient, environmentally sustainable, and renewable alternative for replacing fossil fuels.