It is well-established that neurodegenerative diseases lead to structural and functional reconfigurations in neural networks. However, it is less clear how altered network dynamics contribute to increased vulnerability of specific neuronal populations to disease onset and spread. Here, we aimed to identify how ALS pathology affects the structural and functional dynamics of motor neuron networks, and the underlying biological pathways that contribute to cause such dysfunction. Multielectrode arrays (MEAs) enable longitudinal assessment of electrophysiological activity in vitro, thus facilitating the study of network-wide disruptions associated with neurodegenerative processes. We examined over a period of six weeks the structural and functional development of ALS patient-derived motor neuron networks and healthy control networks. Systematic recordings revealed specific time points for the emergence of potentially maladaptive neural network behavior in the ALS networks. Specifically, the ALS networks exhibited decreased firing rate compared to controls, a lower spike amplitude, reduced bursting, but a higher overall synchrony in network activity. These changes coincided with structural alterations within the networks, as well as with dysregulated expression of genes involved in synapse development and maintenance, cell adhesion and neuroplasticity, as revealed by mRNA sequencing. Overall, our findings suggest that ALS-predisposing mutations can lead to inherent vulnerabilities in motor neurons in terms of impaired synapses, resulting in compensatory mechanisms at the network level. Such compensation could be highly energy demanding and render the network particularly vulnerable to insults, ultimately leading to network degeneration.