Transmission’s efficiency of agricultural tractor is assured by a multipurpose UTTO lubricants (Universal Tractor Transmission Oil). Conventional UTTOs are based on mineral oils, characterized by excellent lubricating properties, but also by low biodegradability and high toxicity, making them a significant source of pollution for environments (soil, water, air). This article reports the results of a study carried out on an experimental bio-based UTTO lubricant as a possible replacement for conventional fluids, with the aim of increasing the sustainability of tractor-based operations. The study was based on endurance tests carried out using an experimental oil test bench that simulates the tractor’s transmission and hydraulic system works, simultaneously applying severe mechanical and hydraulic work cycles. By controlling the pre-set working conditions, it is possible to accelerate the aging of the fluid, and both monitor technical performance (operating pressure and temperature, flowrate, hydraulic and mechanical power transmitted) and the evolution of the chemical-physical characteristics (kinematic viscosity, viscosity index, acidity), indicating its lubricating properties. The bio-based UTTO and a conventional UTTO as a benchmark underwent 150 h work cycle. The comparison of results showed that the two fluids behaved similarly concerning the mentioned parameters, indicating good perspectives for the bio-based one to be introduced in agricultural tractors.