We report the influence of vacancy point defects on the conductivity and piezoelectricity of hydroxyapatite (HAp) nanofibers. A combination of experimental techniques, including conductive atomic force microscopy, electrostatic force microscopy, and switching spectroscopy piezoresponse force microscopy, along with computational modeling, was employed to elucidate the conduction mechanisms and charge accumulation effects in HAp. Our findings demonstrate that oxygen and calcium vacancy defects play a crucial role in the conduction mechanism of HAp nanofibers, specifically through charge-trapping and detrapping processes, as well as in charge accumulation and the piezoelectric response. The Poole-Frenkel conduction mechanism was confirmed by fitting experimental current-voltage data to a theoretical model, revealing a dielectric constant consistent with previously reported theoretical values. These insights contribute to a deeper understanding of the role of point defects in the electrical and piezoelectric properties of HAp, which is essential for optimizing its performance in biomedical applications.