Surface charge and in vitro corrosion resistance are some of the key parameters characterizing biomaterials in the interaction of the implant with the biological environment. Hence, this work investigates the in vitro bioelectrochemical behavior of newly developed oxide nanotubes (ONTs) layers of second-generation (2G) on a Ti–13Zr–13Nb alloy. The 2G ONTs were produced by anodization in 1 M (NH4)2SO4 solution with 2 wt.% of NH4F. The physical and chemical properties of the obtained bamboo-inspired 2G ONTs were characterized using scanning electron microscopy with field emission and energy dispersive spectroscopy. Zeta potential measurements for the examined materials were carried out using an electrokinetic analyzer in aqueous electrolytes of potassium chloride, phosphate-buffered saline and artificial blood. It was found that the electrolyte type and the ionic strength affect the bioelectrochemical properties of 2G ONTs layers. Open circuit potential and anodic polarization curve results proved the influence of anodizing on the improvement of in vitro corrosion resistance of the Ti–13Zr–13Nb alloy in PBS solution. The anodizing conditions used can be proposed for the production of long-term implants, which are not susceptible to pitting corrosion up to 9.4 V.