Ti-6Al-4V with its ELI variant is a potential biomaterial for structural implants. However, its poor resistance to plastic shearing results in poor tribological properties that hinders its widespread use in biomedical implants. Cryogenic treatment being an established technique for improving wear resistance and other mechanical properties of ferrous materials is applied on Ti-6Al-4V ELI alloy. To ensure comprehensive investigations, the widely studied microstructures of Ti-6Al-4V ELI, i.e., lamellar, martensite, two variants of bimodal and mill annealed are considered in this work. Deep cryogenic treatment at the soaking temperature (− 196 °C) for 24 h soaking time followed by tempering has been applied. The impact of cryogenic treatment has been assessed in terms of changes in the mechanical behavior including wear resistance, hardness, tensile properties and impact strength. The alterations in mechanical behavior due to cryogenic treatment are correlated with microstructure evolution. Cryogenic treatment has caused significant improvement in mechanical behavior of Ti-6Al-4V ELI alloy.