An increasing interest in bio-hybrid systems and cell-material interactions is evident in the last years. This leads towards the development of new nano-structured devices and the assessment of their biocompatibility. In the present study, the development of free-standing single layer poly(lactic acid) (PLA) ultra-thin films is described, together with the analysis of topography and roughness properties. The biocompatibility of the PLA films has been tested in vitro, by seeding C2C12 skeletal muscle cells, and thus assessing cells shape, density and viability after 24, 48 and 72 h. The results show that free-standing flexible PLA nanofilms represent a good matrix for C2C12 cells adhesion, spreading and proliferation. Early differentiation into myotubes is also allowed. The biocompatibility of the novel ultra-thin films as substrates for cell growth promotes their application in the fields of regenerative medicine, muscle tissue engineering, drug delivery, and-in general-in the field of bio-hybrid devices.