The neuroengineering of devices capable of interfacing with the central nervous system (CNS) is a growing interdisciplinary area faced with many challenges at the (sub)cellular level, due to the unique anatomy and physiology of the CNS. Arrays of microelectrodes capable of functional extracellular electrical recording and stimulation have recently found application in (pre-)clinical studies, attempting to restore function of damaged neuronal tissue, though use has thus far been limited. Carbon-based nanomaterials have emerged as a promising biocompatible material, capable of forming an excellent microelectrode material through doping with boron. Nanocrystalline diamond (NCD), in particular, demonstrates unique electrical, chemical, thermal and mechanical properties, along with stability, exemplifying its potential as a platform for neuronal interfacing. In this work, we considered NCD as a substrate for ex vivo functional development of mammalian primary neuronal networks. Our main goal was to integrate the advantages that diamond films provide for biological experiments with the large area diamond film technology andto use microelectronic compatible treatment strategies enabling neuronal adhesion and electrophysiological coupling of neurons to NCD substrates. Herein, we describe the successful optimization of diamond surfaces through the use of nanometer-thin Polyethyleneimine coatings, as well as the preparation and functionalization of diamond microelectrode arrays (MEAs), for extended temporal interfacing of NCD to neurons.