The development of surface modification technique has been the subject of the studies regarding the fatigue performance and biological characterization of the modified layers. In the present research work, powder mixed electric discharge machining (PMEDM) a novel nonconventional machining technique has been proposed for surface modification of β-Ti implant for orthopedics application. The surface topography and morphology like roughness, surface cracks, and recast layer thickness of each of the machined specimens were investigated using Mitutoyo surface roughness tester and field-emission scanning electron microscopy (FE-SEM), respectively. This study aims to investigate the effect of surface characteristics of PMEDM process on the fatigue performance and bioactivity of β-Ti implants and moreover a comparative analysis is made on the fatigue performance and biological activity of specimens machined with presently used machining methods like electric discharge machining (EDM) and mechanical polishing. The high cycle fatigue (HCF) performance of polished specimens was superior and had no adverse effect of microstructure on fatigue endurance. As expected, the fatigue behavior of β-Ti implant-based alloy, after undergoing EDM treatment, is poorly observed due to the microrough surface. The fatigue performance is dependent on microstructure and surface roughness of the specimens. Subsequent PMEDM process significantly improves the fatigue endurance of β-Ti implant-based alloy specimens. PMEDMed surface with micro-, sub-micro-, and nano-structured topography exhibited excellent bioactivity and improved biocompatibility. PMEDMed surface enabled better adhesion and growth of MG-63 when compared with the polished and EDMed substrate. Furthermore, the differentiation results indicated that a combination of nanoscale featured submicrorough PMEDMed surface promotes various osteoblast differentiation activities like alkaline phosphatase (ALP) activity, osteocalcin production, the local factor osteoprotegerin, which inhibits osteoclastogenesis.