The influence of novel nanostructured anisotropically electrospun poly(hydroxy butyrate) matrices on skeletal and cardiac muscle-like cell proliferation and differentiation was investigated, in comparison with isotropic and no-topographically cues-provided substrates. After the matrix characterization, in terms of surface SEM imaging and mechanical properties, cell differentiation on the different substrates was evaluated. Myogenin and F-actin staining at several differentiation time-points suggested that aligned nanofibers promote differentiation of both cell types. Moreover, quantitative parameters for each cell line are provided to clarify which aspects of the differentiation process are influenced by the different matrix topographies.