Regenerative cardiac tissue is a promising field of study with translational potential as a therapeutic option for myocardial repair after injury, however, poor electrical and contractile function has limited translational utility. Emerging research suggests scaffolds that recapitulate the structure of the native myocardium improve physiological function. Engineered cardiac constructs with anisotropic extracellular architecture demonstrate improved tissue contractility, signaling synchronicity, and cellular organization when compared to constructs with reduced architectural order. The complexity of scaffold fabrication, however, limits isolated variation of individual structural and mechanical characteristics. Thus, the isolated impact of scaffold macroarchitecture on tissue function is poorly understood. Here, we produce isotropic and aligned collagen scaffolds seeded with embryonic stem cell derived cardiomyocytes (hESC-CM) while conserving all confounding physio-mechanical features to independently assess the effects of macroarchitecture on tissue function. We quantified spatiotemporal tissue function through calcium signaling and contractile strain. We further examined intercellular organization and intracellular development. Aligned tissue constructs facilitated improved signaling synchronicity and directional contractility as well as dictated uniform cellular alignment. Cells on aligned constructs also displayed phenotypic and genetic markers of increased maturity. Our results isolate the influence of scaffold macrostructure on tissue function and inform the design of optimized cardiac tissue for regenerative and model medical systems.