Flexible polyurethane foam (PUF) is a vital material across diverse applications, and its global market is projected to continue growing. Driven by regulatory and consumer demand for sustainable materials, the PUF industry is exploring alternatives to petroleum-derived raw materials, such as vegetable oil-derived bio-polyols. Although bio-based alternatives to fossil-derived foams have been developed, their environmental benefits remain to be fully assessed. Therefore, this study evaluates the environmental performance of flexible PUF production by comparing a conventional fossil-based formulation with a bio-based alternative using a cradle-to-gate Life Cycle Assessment (LCA). The bio-based PUF reduced global warming (6%), fossil resource scarcity (9%), and mineral resource scarcity (6%), but caused significant increases in freshwater eutrophication (91%) and marine eutrophication (19%), mainly due to agricultural processes associated with soybean cultivation. Regardless of the formulation, polyol and toluene diisocyanate production were identified as major environmental hotspots. These results highlight both the decarbonization potential and the trade-offs of bio-based raw materials, underlining the complexity of achieving sustainable PUF production. Overall, the findings provide quantitative insights to guide more sustainable design and sourcing strategies for flexible PUF in the transition from fossil to renewable feedstocks.