Polymer-based nanocomposites loaded with metal nanoparticles are an interesting alternative for the development of antimicrobial food packaging. However, affinity and dispersion of metal nanoparticles could be improved using nanoclays. Therefore, nanocomposite films based on poly(lactic acid) (PLA) and copper modified-montmorillonite were developed by melt-extrusion technique. Copper-modified montmorillonite at different valence states (MtCu(2+) and MtCu(0)) were prepared. The nanocomposites were evaluated by means of their structural, optical, thermal and antimicrobial properties. Results showed that both nanoclays were intercalated in PLA but MtCu(2+) was better dispersed. Therefore, color change Delta E < 2, higher thermal stability and higher elongation at break was evidenced for MtCu(2+)/PLA in comparison to pure PLA. Contrary, MtCu(0) showed the displacement of Cu-0 nanoparticles to the polymer matrix. These free nanoparticles were agglomerated and they leaded on a Delta E > 9 for MtCu(0)/PLA nanocomposites compared with PLA. Moreover, Cu-0 nanoparticles in the matrix accelerated the thermal degradation and decreased the ductility of nanocomposites. On the other hand, both nanocomposites were able to reduce over 2 log(10) of Escherichia coli growth and MtCu(2+)/PLA showed a higher antimicrobial activity against Listeria innocua. These results showed the potential of nanocomposite films based on PLA and copper-modified montmorillonite for antimicrobial food packaging applications.