Nanofibers can be produced by various techniques, such as a broad range of electrospinning techniques to produce nanofiber mats from different polymers or polymer blends, often filled with metallic or semiconducting nanoparticles or by different nanotechnological bottom-up or top-down methods. They are important parts of a wide variety of energy applications, such as batteries, fuel cells, photovoltaics, or hydrogen storage materials. Usually, their physical or chemical parameters are measured by averaging over a fiber bundle or a part of a nanofiber mat. Here, we report the possibility of measuring the different physical and chemical properties of single nanofibers and nanowires. Such measurements of single nanofiber properties are more complicated than investigations of fiber bundles or whole nanofiber mats and, thus, are less often found in the literature. After a fast increase in such investigations between 2001 and 2009, the numbers of respective studies are now stagnating. This review thus aims to make the different possibilities more visible to a broader scientific audience by providing several examples based on atomic force microscopy (AFM) and other broadly available techniques. The focus of this review is on technologies that reveal more information than the pure surface morphology of nanofibers or nanowires, such as mechanical properties or wettability, porosity, or electrical conductivity.