As undoped diamond seed nanoparticles remain non-conductive after the growth of B-doped diamond films, they represent localized regions of low conductivity and basically form a resistive barrier at the diamond film–substrate interface. This can be detrimental for diamond applications that require a good electrical contact with the substrate, such as small electrical probes and electrodes. Therefore, the use of B-doped seeds is highly required as it can result in a well-conductive interfacial layer. In this study, we show by electrically probing the diamond interfacial layer with nanometer-scale resolution using scanning spreading resistance microscopy (SSRM) that B-doped seeds indeed lead to highly conductive regions at the interface and, in our case, exhibit a resistance similar to the surrounding highly B-doped grown diamond. We conclude that a seed layer that consists of B-doped diamond nanoparticles will allow the formation of diamond films with a well-conductive interfacial layer.