This paper aims to demonstrate the possibility of exploiting poly-L-lactic acid (PLLA) ultra-thin films as platforms for bio-hybrid actuation. Firstly, flat PLLA nanofilms at different concentrations (15 and 25 mg/ml in dichloromethane) were tested with contractile cardiomyocytes. The results obtained using motion vector analysis, a non-invasive method capable of estimating flow velocities on recorded videos, demonstrated that PLLA nanofilms were able to move under the contraction of muscle cells. Immunofluorescence images reflected good cell spreading, thus confirming that these films are promising matrices for bio-hybrid actuation. Subsequently, microgrooved PLLA nanofilms were fabricated, in order to drive muscle cell distribution on an anisotropic surface, thus optimizing the system's efficiency. After matrix characterization, in terms of AFM and SEM imaging, we investigated the viability and morphology of C2C12 skeletal muscle cells (a more controllable muscle cell type), 24 h after cell seeding as well as at the 7-day differentiation state.