The medical field is continuously seeking new solutions and materials, where cellulose materials due to their high biocompatibility have great potential. Here we investigate the applicability of cellulose acetate (CA) electrospun fibers for bone tissue regeneration. For the first time we show the piezoelectric properties of electrospun CA fibers via high voltage switching spectroscopy piezoresponse force microscopy (HVSS-PFM) tests, which are followed by surface potential studies using Kelvin probe force microscopy (KPFM) and zeta potential measurements. Piezoelectric coefficient for CA fibers of 6.68 ± 1.70 pmV-1 along with high surface (718 mV) and zeta (-12.2 mV) potentials allowed us to mimic natural electrical environment favoring bone cell attachment and growth. Importantly, the synergy between increased surface potential and highly developed structure of the fibrous scaffold led to the formation of a vast 3D network of collagen produced by osteoblasts only after 7 days of in vitro culture. We clearly show the advantages of CA scaffolds as a bone replacement material, when long-lasting structural support is needed.