Carbon nanofibers (CNFs) have applications in a wide range of technological and scientific fields. The connections between their micro- and macrostructure and observed performance are, however, currently lacking. This hinders the realization of their full potential. In this paper, we correlate the microstructure of CNFs grown on two types of substrates: (1) Si + 20 nm Ti + 20 nm Ni, and (2) Si + 80 nm Cr + 20 nm Ni, to their surface chemistry. We use transmission electron microscopy (TEM), supported by energy dispersive X-ray spectroscopy (EDS) and X-ray diffraction (XRD) analysis, to describe the morphology and structure of CNFs as well as the underlying interfacial layers. Then, we study the similarities and differences in chemistry of these two types of CNFs using X-ray photoelectron spectroscopy (XPS) and X-ray absorption spectroscopy (XAS) and correlate them with the observed structural features of the fibers. Vertically aligned, tip-type fiber growth was observed on both substrates. TEM micrographs show that the CNFs grown on the Cr + Ni substrates have a slightly distorted herringbone-like structure, whereas fibers grown on the Ti + Ni substrates have relatively ill-defined structure with basal planes pointing outwards. Consequently, the latter possess a richer surface chemistry, which is apparent from the wider peaks and more spectral features observed during XAS and XPS measurements. This analysis provides us with some of the missing structure-chemistry connections, which can subsequently be expanded towards including correlations of these features with observed performance of the CNFs in different applications. Ultimately, this enables us to tailor features of the CNFs for specific target fields.