Given the current EU decarbonization targets, the railway transport is a key player to boost mobility toward more sustainable transportation, as it is currently the cleanest high-volume mode of locomotion available. However, a study analyzing the life cycle environmental impact of the existing conventional Portuguese railway has never been performed. Aiming to address this research gap, this paper presents an attributional life cycle assessment (LCA) to quantify the environmental impacts of the Portuguese railway infrastructure and rolling stock, using the Douro line case study. Through the LCA methodology, the current setting (using electric and diesel rolling stock) and three scenarios of full-line electrification (considering 2019, 2030, and 2050 electricity mixes) were analyzed for hotspot identification and an outlook on EU-aligned long-term sustainability prospects. In the current scenario, railway operation accounts for 74% of the total carbon footprint, mostly due to the fuel use of diesel trains and the expended electricity of electric train and infrastructure operation. The total electrification of the line and rolling stock can reduce carbon emissions by 38%, 56%, and 63%, if the 2019, 2030, and 2050 electricity mixes are considered, respectively. Further reductions could also be achieved with on-site renewable energy generation and through future low-carbon construction work strategies.