Agriculture may hold the key to a sustainable future. By efficiently capturing atmospheric CO2, we can simultaneously produce food, feed, biomass, and biofuels. For more eco-friendly soil processing practices, biofuels can replace diesel in agricultural machinery, significantly reducing the carbon footprint of crop production. Thus, biofuel production can be a sustainable solution for a future with a decreasing carbon footprint. This paper examines the possibility of replacing petroleum-based fuels with 100% biofuels to continue powering heavy-duty vehicles, where the use of electric vehicles is not the optimal solution. This study particularly focused on the operating scenario of heavy-duty engines under medium to high loads, typical of transport or soil processing in agriculture. Diesel was used as a benchmark, and each alternative, such as vegetable oil, methyl ester (B100), and methyl ester–ethanol blends (90B10E, 80B20E, and 70B30E), was tested individually. To find a sustainable fuel substitute, the goal was to identify a biofuel with a kinematic viscosity similar to that of diesel for a comparable spray process. Experimental results showed that an 80% methyl ester and 20% ethanol blend had a kinematic viscosity close to that of diesel. In addition to diesel, this blend resulted in a 48.6% reduction in exhaust gas opacity and a 6.54% lower specific fuel consumption (BSEC). The main aim of the tests was to find a 100% biofuel substitute without modifying the fuel injection systems of existing engines.