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The transformation of transport systems towards the use of alternative fuels is a key element in the strategy to reduce greenhouse gas emissions and achieve climate neutrality. This paper presents a life cycle assessment (LCA) of selected alternative fuels used in public transport, including biofuels, electricity, hydrogen, and synthetic fuels, based on available literature data. It outlines the methodological framework of LCA in accordance with ISO 14040/44 standards, with a particular focus on defining the goal and scope of the analysis, selecting the functional unit, establishing system boundaries, and developing life cycle inventory (LCI). Various methods for assessing environmental impacts are considered to identify environmental burdens at different stages of the fuel life cycle – from raw material extraction, through production, distribution, to end use. The comparative analysis highlights significant differences in results depending on the origin of raw materials, production technologies, and energy mix. The paper identifies the key life cycle stages that most significantly determine total greenhouse gas emissions, thereby enabling the identification of the most effective emission reduction pathways in public transport. The findings indicate that the environmental benefits of alternative fuels are highly context-dependent and require comprehensive assessment across the entire life cycle.