The microstructure of lubricating greases greatly conditions their in-service performance. In that sense, optimal testing protocols are required in order to accomplish their correct morphological characterization. This study explores and compares the suitability of atomic force microscopy (AFM) and scanning electron microscopy (SEM) techniques for imaging six different commercial metallic soap-based greases and two novel biopolymer-based formulations. Pros and cons of both techniques and the effect of sample preparation protocol were analysed. The results revealed a wide variety of morphological characteristics depending on composition. Thus, the four anhydrous calcium-based greases demonstrated two clearly distinct microstructures (fibrous and granular) determined by the type of base oil employed. With regard to the lithium complex greases, the typically reported microstructure characterized by well-defined entangled and fibrous network was observed in both AFM and SEM techniques. As for the two biopolymer-based greases, fibre networks were also encountered. Besides this, selected greases were subjected to different tribological tests, and the effect of high-shear frictional working treatments on their microstructure was also analysed. As a result of the friction and internal wear, the AFM results evidenced microstructural changes which depended on grease composition. Overall, the combined use of AFM and SEM techniques was demonstrated to be a powerful approach to microstructurally characterize lubricating greases.