Collagen is a widely used biomaterial in cardiac tissue engineering studies. However, as a natural material, it suffers from variability between batches that can complicate the standardization of culture conditions. In contrast, synthetic materials are modifiable, have well-defined structures and more homogeneous batches can be produced. In this study, several collagen-like synthetic self-assembling nanofiber hydrogels were examined for their suitability for cardiomyocyte culture in 2D and 3D. Six different nanofiber coatings were used in the 2D format with neonatal rat cardiomyocytes (NRCs) and human embryonic stem-cell-derived cardiomyocytes (hESC-CMs). The viability, growth, and functionality of the 2D-cultured cardiomyocytes were evaluated. The best-performing nanofiber coatings were selected for 3D experiments. Hydrophilic pH-sensitive nanofiber hydrogel coassembled with hyaluronic acid performed best with both NRCs and hESC-CMs. Hydrophilic non-pH-sensitive nanofiber hydrogels supported the growth of NRCs; however, their ability to promote attachment and growth of hESC-CMs was limited. NRCs also grew on hydrophobic nanofiber hydrogels; however, the cell-supporting capacity of these hydrogels was inferior to that of the hydrophilic hydrogel materials. This is the first study demonstrating that hydrophilic self-assembling nanofiber hydrogels support the culture of both NRCs and hESC-CMs, which suggests that these biomaterials hold promise for cardiac tissue engineering.