This study investigates the potential of biofuels to mitigate the environmental impact of passenger vessels. Employing a life cycle assessment methodology, this research comprehensively analyzes the environmental footprint of various fuels throughout the life cycle of a passenger vessel, encompassing construction, fuel production, operation, maintenance, and decommissioning. This study demonstrates the application of life cycle impact assessment to evaluate the environmental footprint of a bio-fueled passenger vessel operating on the Baltic Sea route from Tallinn-Helsinki, Helsinki-Åland, and Åland-Stockholm, totaling a round-trip distance of around 700 nautical miles. Ten different fuels, including several biofuel options, were evaluated using two established impact assessment methodologies. This life cycle assessment study focused on five major environmental impact categories: eutrophication, ozone depletion, climate change, and human and eco-toxicity. A comparative analysis encompassed Heavy fuel oil, bio-hydrotreated vegetable oil, bio-dimethyl ether, marine diesel oil, biodiesel, liquefied natural gas, methanol, bio-methanol, ethanol, and bio-ethanol. Based on impact categories like Global warming potential (GWP 100) and Global temperature potential (GTP 100), the findings suggest significantly lower environmental impacts of biofuels than all other fuel options. Notably, biofuels significantly reduced environmental impact, ranging from 70 % to 90 % per tonne-kilometer across most categories. These results highlight the promise of biofuels as a sustainable alternative fuel source for the maritime transportation sector, potentially reducing their environmental footprints significantly. In summary, biofuels offer a pragmatic and timely approach to enhancing passenger vessels' sustainability and operational efficiency amidst the maritime sector's transition towards a cleaner future.