The goal of this study is to investigate the influence of different types of modifiers, such as sodium hyaluronate (NaH), graphene oxide (GO), silica oxycarbide (SiOC) and oxidation process (ox) on physicochemical, morphological, and biological properties of electrospun carbon nanofibers (eCNFs). Scanning electron microscopy, X-ray photoelectron spectroscopy and infrared spectroscopy (FTIR) were used to evaluate the microstructure and chemistry of as-prepared and modified CNFs. The electrical properties of CNFs scaffolds were examined using a four-point probe method to evaluate the influence of modifiers on the volume conductivity and surface resistivity of the obtained samples. The wettability of the surfaces of modified and unmodified CNFs scaffolds was also tested by contact angle measurement. During the in vitro study all samples were put into direct contact with human chondrocyte CHON-001 cells and human osteosarcoma MG-63 cells. Their viability was analysed after 72 h in culture. Moreover, the cell morphology and cell area in contact with CNFs was observed by means of fluorescence microscopy. The obtained results show great potential for the modification of CNFs with polymer, ceramic and carbon modifiers, which do not change the fiber form of the substrate but significantly affect their surface and volume properties. Preliminary biological studies have shown that the type of modification of CNFs affects either the rate of increase in the number of cells or the degree of spreading in relation to the unmodified sample. More hydrophilic and low electrically conductive samples such as CNF_ox and CNF_NaH significantly increase cell proliferation, while other GO and SiOC modified samples have an effect on cell adhesion and thus cell spreading. From the point of view of further research and the possibility of combining the electrical properties of modified CNF scaffolds with electrical stimulation, where these scaffolds would be able to transport electrical signals to cells and thus affect cell adhesion, spreading, and consequently tissue regeneration, samples CNF_GO and CNF_SiOC would be the most desirable.