In microbial oceanography, cell size, volume and carbon (C) content of pelagic bacteria and archaea ('bacteria') are critical parameters in addressing the in situ physiology and functions of bacteria, and their role in the food web and C cycle. However, because of the diminutive size of most pelagic bacteria and errors caused by sample fixation and processing, an accurate measurement of the size and volume has been challenging. We used atomic force microscopy (AFM) to obtain high-resolution images of pelagic bacteria and Synechococcus. We measured the length, width and height of live and formalin-fixed pelagic bacteria, and computed individual cell volumes. AFM-based measurements were compared with those by epifluorescence microscopy (EFM) using 4',6-diamidino-2-phenylindole (DAPI). The ability to measure cell height by AFM provides methodological advantage and ecophysiological insight. For the samples examined, EFM (DAPI)-based average cell volume was in good agreement (1.1-fold) with live sample AFM. However, the agreement may be a fortuitous balance between cell shrinkage due to fixation/drying (threefold) and Z-overestimation (as EFM does not account for cell flattening caused by sample processing and assumes that height=width). The two methods showed major differences in cell volume and cell C frequency distributions. This study refines the methodology for quantifying bacteria-mediated C fluxes and the role of bacteria in marine ecosystems, and suggests the potential of AFM for individual cell physiological interrogations in natural marine assemblages.