In recent years, bio-lubricants have received a growing interest for industrial applications. Still, a full-scale implementation in machinery lubrication requires a thorough evaluation of their performance through tribological and operational tests to stand upon their performance. Additionally, the promising outcomes achieved by nanoadditives in improving the performance of synthetic lubricants have prompted research efforts to identify suitable nanoadditives for bio-grease. This paper introduces a bio-grease from a hybrid vegetable oil and glycerol monostearate as a thickener for the lubrication of rolling bearings. Activated carbon nanoparticles (ACNPs) as nanoadditives were synthesized, characterized, and incorporated into the bio-grease at concentrations of 0.5, 1, and 2% by weight. Tribo-tests were conducted on these bio-grease blends, and running tests were carried out using 6006 ball bearings on a custom test rig. Throughout a 30-min test run under a radial load of 10% of the bearing’s dynamic load rating, mechanical vibrations and power consumption were measured and analyzed for each bearing. The bio-grease with ACNPs exhibited a substantial reduction in wear scar diameter (WSD) and coefficient of friction (COF), achieving improvements of up to 73.6 and 65%, respectively, in comparison to lithium grease. Furthermore, the load carrying capacity was enhanced by 200%. The study revealed a strong correlation between measured vibration amplitudes and the viscosity of the bio-grease. The absence of high frequency resonant bands in vibration spectra indicated that the test grease samples satisfied the conditions of elastohydrodynamic lubrication, and these findings were corroborated through calculations of the minimum oil film thickness.