Disinfection of electrospun materials is commonly carried out by ethanol (EtOH) soaking. However, in the literature no systematic investigation on the effect of EtOH disinfection on scaffold properties is available. In this study electrospun poly(l-lactic acid) (PLLA) scaffolds were differently treated in EtOH and changes of fiber morphology, fiber surface topography and scaffold thermo-mechanical properties were evaluated. It was found that EtOH induced the formation of an amount of crystal phase, in the initially amorphous fibers, which depends on EtOH grade, soaking time and temperature. Completely amorphous PLLA (a-PLLA) and semicrystalline PLLA (sc-PLLA) scaffolds were produced by applying different EtOH treatments to as-spun PLLA scaffold. Compared to a-PLLA, sc-PLLA was stiffer and composed of fibers with rougher surface. Cell culture performed by using permanent cell line H9c2 demonstrated that changes of scaffold properties determined a different cell response, in particular in the expression of proteins correlated to cell motility and cell adhesion.