In this work, the experimental investigation of the surface integrity and biomechanical properties of the superficial layer obtained by wire electrical discharge machining (W-EDM) of Ti-6Al-4V alloy for biomedical application has been carried out. The surface morphology and elemental composition of the superficial layer have been investigated by field-emission scanning electron microscope (FE-SEM) and energy dispersive X-ray spectroscopy (EDS) techniques. The micro-mechanical behavior in terms of compressive strength and surface hardness was studied using the micro-pillar and nano-indentation technique. The corrosion resistance and in vitro bioactivity have been investigated using electrochemical and immersion test. Morphological analysis showed that surface morphology and superficial layer thickness were affected by peak current, pulse-duration and pulse-interval. The niobium (Nb)-rich layer was developed in superficial layer zone. The low peak current (3–6[Formula: see text]A), low pulse-duration (5–10[Formula: see text][Formula: see text]s) and high pulse-interval ([Formula: see text]s) have been recommended for better surface morphology and thin superficial layer (ranging from 4–6[Formula: see text][Formula: see text]m) free from surface defects. The micro-pillar and nano-indentation results showed that the superficial layer comprised of a brittle structure that improved the mechanical properties of the layer and the compressive strength was measured to be 1198 MPa. The corrosion resistance analysis revealed that the Nb-rich layer in the superficial layer improved the corrosion resistance and bioactivity. Excellent apatite growth has been found in the W-EDM-processed zone. The W-EDM can be used for the biomedical industry as a potential surface engineering technique.