In vivo, multiple biophysical cues provided by highly ordered connective tissues of the extracellular matrix regulate skeletal muscle cells to align in parallel with one another. However, in routine in vitro cell culture environments, these key factors are often missing, which leads to changes in cell behavior. Here, we present a simple strategy for using optical media discs with nanogrooves and other polymer-based substrates nanomolded from the discs to directly culture muscle cells to study their response to the effect of biophysical cues such as nanotopography and substrate stiffness. We extend the range of study of biophysical cues for myoblasts by showing that they can sense ripple sizes as small as a 100 nm width and a 20 nm depth for myotube alignment, which has not been reported previously. The results revealed that nanotopography and substrate stiffness regulated myoblast proliferation and morphology independently, with nanotopographical cues showing a higher effect. These biophysical cues also worked synergistically, and their individual effects on cells were additive; i.e., by comparing cells grown on different polymer-based substrates (with and without nanogrooves), the cell proliferation rate could be reduced by as much as ~29%, and the elongation rate could be increased as much as ~116%. Moreover, during myogenesis, muscle cells actively responded to nanotopography and consistently showed increases in fusion and maturation indices of ~28% and ~21%, respectively. Finally, under electrical stimulation, the contraction amplitude of well-aligned myotubes was found to be almost 3 times greater than that for the cells on a smooth surface, regardless of the substrate stiffness.