The tribological properties of lubricants can be effectively improved by the introduction of amphiphilic molecules, whose performance is largely affected by their polar head groups. In this work, the tribological performance in steel–steel contacts of two isomers, glycerol monostearate (GMS) and stearyl glycerate (SG), a glyceride and a glycerate, were investigated as organic friction modifiers (OFM) in hexadecane. SG exhibits a much lower friction coefficient and wear than GMS despite their similar structures. The same applies when comparing the performance of oleyl glycerate (OG) and its isomer, glycerol monooleate (GMO). Surface chemical analysis shows that SG forms a polar, carbon-based, tribofilm of around tens of nanometers thick, while GMS does not. This tribofilm shows low friction and robustness under nanotribology test, which may contribute to its superior performance at the macro-scale. The reason for this tribofilm formation can be due to the stronger adsorption of SG on the steel surface than that of GMS. The tribofilm formation can be stress-activated since lower friction and higher tribofilm coverage can be obtained under high load. This work offers insights into the lubrication mechanism of a novel OFM and provides strategies for OFM design.