Neuronal networks are surrounded by the extracellular matrix (ECM), which functions both as a scaffold and as a regulator of neuronal function. The ECM is in turn dynamically altered through the action of serine proteases, which break down its constituents. This pathway has been implicated in the regulation of synaptic plasticity and intrinsic excitability. Here, we determined the effects of acutely inhibiting two important regulators of the ECM, Urokinase Plasminogen Activator and Neuropsin, selectively and potently with the inhibitor UAMC-01162. Spontaneous electrophysiological activity was recorded from in vitro primary rat cortical cultures using microelectrode arrays. While inhibition at a low dosage had no significant effect, at elevated concentrations network bursting dynamics and functional connectivity were drastically altered. These results indicate that serine protease inhibition affects neuronal and synaptic properties, likely through their actions on the ECM. We propose that in the acute phase, a transient increase of excitatory synaptic efficacy is compensated for by a downregulation of single-cell excitability.