Organophosphorus compounds (OPs) are pesticides of worldwide use due to the acute insecticidal effects mediated by the inhibition of esterases of the central nervous system (mainly acetylcholinesterase and neuropathy target esterase (NTE)). OPs need to inhibit acetylcholinesterase to be effective insecticides, but not NTE since its inhibition might cause progressive, irreversible delayed neuropathy in humans and other species. Additionally, other neurological and neurodevelopmental toxic effects with unknown targets have been reported in humans or animals chronically exposed to OPs. We used a mixed neuronal/glia culture derived from well-characterised human embryonal carcinoma stem cells (hNT2) to determine if neuropathic OP mipafox and non-neuropathic OP paraoxon are able to alter the neuronal differentiation process evaluated by gene expression studies, neuronal electrical activity measurements and neural cell morphology quantification. Exposure to paraoxon at non-cytotoxic concentrations altered the expression of different genes involved mainly in signalling pathways related to chromatin assembly and nucleosome integrity, generating cultures with a larger number of differentiated neurons-like cells and branching points than in the control. Moreover, these paraoxon-exposed neuronal-like cells displayed reduced electrical activity when compared with the control neurons as measured by Micro Electrode Array Chips. Similarly, exposure to mipafox, a known inhibitor of NTE activity, also reduced the electrical activity of hNT2 cultures differentiated into neurons-like cells, but no significant changes in cell morphology or gene expression were detected. Therefore, we conclude that paraoxon is able to strongly disturb in vitro neurodifferentiation, while the absence of morphological and transcriptional alterations did not allow us to conclude if the electrophysiological alterations detected in mipafox-exposed neurons are due to neurodevelopmental toxicity or to effects on mature neurons.