Scanning tunneling and atomic force microscopies (STM/AFM) have been employed to investigate the initial stages of the surface modification of highly oriented pyrolytic graphite (HOPG) by oxygen plasma. Through a rigorous control of very short exposure times to the plasma it was determined, by means of STM, that the attack starts with the formation of single-atom vacancies (detected as 1 nm wide protrusions) randomly distributed all over the basal planes. The mobility of the active species from the plasma adsorbed on the basal planes was evidenced at following stages by the Marked tendency of HOPG to develop multiatom vacancies and was estimated to be on the order of tens of nanometers. The multiatom vacancies appeared as large (up to 5 nm in diameter) protrusions in the STM images, although it could be established that their actual size (the physical region with missing carbon atoms) was not above 1 nm. The fact that very large atomic vacancies were developed by the present plasma treatments made it possible to study the physicochemical properties of this type of defect by AFM for the first time. Lateral force images obtained in the contact mode displayed enhanced friction due to the vacancies, the origin of which is discussed. Likewise, the structural and chemical heterogeneity of the defects could be revealed independently of one another by phase imaging in the tapping mode and choosing appropriate tip-sample interaction regimes. These results shed some light into the possibility of visualizing active sites on the surface of carbon materials at very high resolution, a question of scientific and technological relevance.