The effect of the post-annealing process on different properties of poly (L-lactic acid) (PLLA) nanofibers has been investigated in view of their use in energy-harvesting devices. Polymeric PLLA nanofibers were prepared by using electrospinning and then were thermally treated above their glass transition. A detailed comparison between as-spun (amorphous) and annealed (semi-crystalline) samples was performed in terms of the crystallinity, morphology and mechanical as well as piezoelectric properties using a multi-technique approach combining DSC, XRD, FTIR, and AFM measurements. A significant increase in the crystallinity of PLLA nanofibers has been observed after the post-annealing process, together with a major improvement of the mechanical and piezoelectric properties.