There is a great need for acellular, fully vascularized, and biocompatible myocardial scaffolds that provide agreeable biological, nutritional, and biomechanical niches for reseeded cells for in vitro and in vivo applications. We generated myocardial flap scaffolds comprising porcine left-anterior ventricular myocardium and its associated coronary arteries and veins and investigated the combinatorial effects of sodium dodecyl sulfate (SDS) and sodium hydroxide (NaOH) perfusion on both the myocardial extracellular matrix (ECM) and the vascular ECM. Results showed that all scaffolds displayed a fully intact and patent vasculature, with arterial burst pressures indistinguishable from native coronary arteries and perfusion to the level of capillaries. Scaffolds were free of cellular proteins and retained collagen and elastin ECM components, exhibited excellent mechanical properties, and were cytocompatible toward relevant seeded cells. SDS perfusion preserved collagen IV, laminin, and fibronectin well, but only reduced DNA content by 33%; however, this was further improved by post-SDS nuclease treatments. By comparison, NaOH was very effective in removing cells and eliminated more than 95% of tissue DNA, but also significantly reduced levels of laminin and fibronectin. Such constructs can be readily trimmed to match the size of the infarct and might be able to functionally integrate within host myocardium and be nourished by direct anastomotic connection with the host's own vasculature; they might also be useful as physiologically accurate models for in vitro studies of cardiac physiology and pathology.