Evaluation of diamond nanoparticles (DNPs) electrical potential under ambient environment is important for their application in electronics as well as sensors and biology. Here we use a novel methodology for characterization of nanoparticles based on recording of their electrical potential as a function of their size by two-pass Kelvin force microscopy (KFM). We study thermally oxidized detonation DNPs of 5 nm nominal size. The nanoparticles were deposited from diluted water solutions on a Si substrate half coated with Au. The KFM using conductive Si tip resolved characteristic negative potential differences of 10–60 mV on nanoparticles versus the substrate. When negative bias voltage to the KFM tip (−4 V) is applied during the topography acquisition (the first pass), subsequent potential measurement (the second pass) shows inversion of nanoparticles potential contrast. The same effects were observed also in the case of 20 nm colloidal Au nanoparticles. This effect is reversible and it is attributed to a charge retention in (or on) the nanoparticles.