In the brain, the neuronal activity is kept and controlled by a precise and well-defined “Excitatory/Inhibitory balance” among neurons, which holds importance in neurodegenerative and neurodevelopmental disorders. With the final aim of generating a reliable in vitro system of the human brain, this work was focused on the interplay between excitatory (E) and inhibitory (I) neurons in neural networks derived from human induced pluripotent stem cells (hiPSCs), deepening the critical role of heterogeneity. We exploited high-density Micro-Electrode Arrays (MEAs, 2304 electrodes) to explore two neuronal culture configurations: 100% excitatory (100E) and 75% excitatory / 25% inhibitory (75E25I) neurons. This allowed to broadly characterize the spontaneous electrophysiological activity of mature neuronal cultures at 56 Days In Vitro, a time point in which the GABA shift has already occurred. We explored the impact of heterogeneity through chemical stimulation. In particular, the administration of BIC led to an increase in terms of firing and bursting activity only in the 75E25I configuration, while APV and CNQX caused substantial alterations on both dynamics and functional connectivity. On the other hand, the electrical stimulation revealed that 100E configuration responded reliably, while the 75E25I required additional parameter tuning for improved responses. Our findings advance the understanding of different neuronal interactions and their role in the network activity, providing insights for potential therapeutic interventions in neurological conditions. Overall, our work contributes to the development of a valuable human in vitro system for investigating physiological and pathological conditions, highlighting the critical role of neuron diversity in neural network dynamics.