: Herein we present the production of novel nanocomposite films consisting of polylactic acid (PLA) polymer and the inclusion of nanoparticles of lignin (LNP), ZnO and hybrid ZnO@LNP (ZnO, 3.5% wt, ICP), characterized by similar regular shapes and different diameter distribution (30–70 nm and 100–150 nm, respectively). The obtained set of binary, ternary and quaternary systems were similar in surface wettability and morphology but different in the tensile performance: while the presence of LNP and ZnO in PLA caused a reduction of elastic modulus, stress and deformation at break, the inclusion of ZnO@LNP increased the stiffness and tensile strength (σb = 65.9 MPa and EYoung = 3030 MPa) with respect to neat PLA (σb = 37.4 MPa and EYoung = 2280 MPa). Neat and nanocomposite PLA-derived films were suitable for adult human bone marrow-mesenchymal stem cells and adipose stem cell cultures, as showed by their viability and behavior comparable to control conditions. Both stem cell types adhered to the films’ surface by vinculin focal adhesion spots and responded to the films’ mechanical properties by orchestrating the F-actin–filamin A interaction. Collectively, our results support the biomedical application of neat- and nanocomposite-PLA films and, based on the absence of toxicity in seeded stem cells, provide a proof of principle of their safety for food packaging purposes.