Perineuronal nets (PNNs) are specialized extracellular matrix structures that surround subsets of neurons throughout the central nervous system (CNS). They are made up of chondroitin sulfate proteoglycans (CSPGs), hyaluronan, tenascin-R, and many other link proteins that together make up their rigid and lattice-like structure. Modulation of PNNs can alter synaptic plasticity and thereby affect learning, memory, and cognition. In the present study, we degraded PNNs in the medial prefrontal (mPFC) and posterior parietal (PPC) cortices of Long–Evans rats using the enzyme chondroitinase ABC (ChABC), which cleaves apart CSPGs. We then measured the consequences of PNN degradation on spatial working memory (WM) with a trial-unique, non-matching-to location (TUNL) automated touchscreen task. All rats were trained with a standard 6 sec delay and 20 sec inter-trial interval (ITI) and then tested under four different conditions: a 6 sec delay, a variable 2 or 6 sec delay, a 2 sec delay with a 1 sec ITI (interference condition), and a 20 sec delay. Rats that received mPFC ChABC treatment initially performed TUNL with higher accuracy, more selection trials completed, and fewer correction trials completed compared to controls in the 20 sec delay condition but did not perform differently from controls in any other condition. Rats that received PPC ChABC treatment did not perform significantly differently from controls in any condition. Posthumous immunohistochemistry confirmed an increase in CSPG degradation products (C4S stain) in the mPFC and PPC following ChABC infusions while WFA staining intensity and parvalbumin positive neuron number were decreased following mPFC, but not PPC, ChABC infusions. These findings suggest that PNNs in the mPFC play a subtle role in spatial WM, but PNNs in the PPC do not. Furthermore, it appears that PNNs in the mPFC are involved in adapting to a challenging novel delay, but that they do not play an essential role in spatial WM function.