Uruguay, a country highly dependent on foreign fossil fuels, recently began developing agro-industrial chains oriented toward renewable bioenergy sources. However, the large number of potential technologies for biofuel production does not provide clear information on production under neotropical conditions or on their potential environmental impacts. Therefore, the National Agricultural Research Institute proposed the first methodological approach for assessing potential agro-industrial chains oriented toward bioenergy production. The effort focused, in the long term, on applying Life Cycle Assessment as the main tool to define sustainability and, in the short term, on estimating the energy return on investment (EROI) and identifying potential feedstocks for biofuel technologies in Uruguay. This project analysed the Uruguayan feedstocks with the highest energy potential (eucalyptus wood, grain sorghum, sweet sorghum, and sweet potato). The energy assessment was based on spreadsheet calculations. The life cycle inventory (LCI) was developed based on agronomic mean data collected from the main forest/agriculture companies and the main Uruguayan agronomist researcher for each crop. Similarly, the average of rate/dosages was obtained for each step. The energy conversion factors utilised were obtained from international publications when primary data were not available in the national survey. The assessment of eucalyptus wood, sweet potato, grain sorghum, and sweet sorghum for bioenergy generation showed EROIs of 0.56, 1.2, 3.5, and 7.2, respectively.