The use of bio-based feedstock for formulation of protective coatings has been implemented in the supply of building blocks synthesized from biomass. Polyurethanes are of great interest due to their wide range of applications such as elastomers, fibers, foams, adhesives, coatings, or sealants. The alternation of traditional polyurethane coatings by bio-based polyurethanes focussed on the replacement of the polyisocyanate component in combination with polyester or polyacrylate polyols. In this research, the performance of an aliphatic isocyanate synthesized from crude oil (i.e., HDI or hexamethylenediisocyanate) has compared to an alternative synthesized through fermentation of biomass (i.e., PDI or pentamethylenediisocyante). As the chemical structure of the bio-based PDI is slightly different with an aliphatic chain of five compared to six carbon atoms, similar or better performance as protective coating is demonstrated. The application of the bio-based PU coatings on steel coupons resulted in lower drying times and higher hardness with similar gloss, chemical resistance and scratch resistance. In particular, the resistance of bio-based coatings after QUV accelerated weathering testing was improved owing to the better hydrophobicity of bio-based polyurethane coatings. There was a gradual trend in evolution of the performance with stepwise replacing fossil-based into bio-based content up to a maximum bio-content of 25% in the coating.