Humans are continuously exposed to a wide variety of chemicals. Animal and bi-dimensional (2D) in vitro models currently represent the gold standard for chemical toxicity assessment, although they do not adequately reproduce the real human scenario, thus resulting in poorly predictive results. In this context, New Approach Methodologies are needed for a proper assessment of chemical toxicity. For instance, three-dimensional (3D) bioengineered in vitro models have the potential to closely represent the human physio-pathological milieu, enabling a more reliable evaluation of chemical toxicity. Among human tissues/organs, the myocardium is a target organ for chemical- and drug-induced toxicity. In this work, a custom-made poly(ester urethane) was synthesized and used as raw material to fabricate a platform of multi-layered structures with different geometrical features through melt extrusion additive manufacturing. The designed matrices were also surface functionalized with proteins to improve their biomimesis of the native cardiac extracellular matrix. The physico-chemical properties of the constructs proved their suitability for the development of in vitro bioengineered cardiac tissue models, enabling the investigation of physio-pathological processes and cardiotoxicity testing.