In this study, Ti6Al4V plates were gone through three steps of pretreatment process (sandblasting + acid etching + preheating) followed by biomimetic coating of pure hydroxyapatites (HAp) on pretreated substrate. Pretreated substrates were examined by SEM, EDS, AFM, FTIR, and XRD; meanwhile, prepared coatings were examined by SEM, EDS, FTIR, XRD, XPS, scratch test, and in vitro cell studies (SaOS-2 cell line). Prepared coatings were also investigated in terms of antibacterial property by determining the survival of the Staphylococcus epidermidis on the surface. Structural analysis results of pretreated substrates proved that surface roughness increased proportionally as sandblasting, acid etching, and preheating process were performed. Successively, surface became more porous according to SEM images which were also supported by AFM results that indicate that Ra increased up to 953 nm values. EDS and FTIR spectra showed that functional groups were formed with the pretreatment procedure. Especially titanium salts were formed during acid etching process while preheating caused oxidation. XRD spectra revealed that as-received substrates mostly consisted of alpha-Ti, acid etching resulted in formation of TiH2 structure and oxidation resulted to form rutile and anatase structure patterns. Pretreatment procedure had positive effect on biomimetic coatings according to the evaluation of structural, mechanical, and biological properties. SEM analysis revealed that HAp nucleation started 4 days after immersion and nucleation increased with the incubation time. EDS results indicated that maximum Ca/P ratio (1.48) was achieved by P14. FTIR, XRD, and XPS analysis proved the presence of hydroxyapatite on the substrate surface. The pretreatment procedure had significant influence on mechanical properties of the coatings resulting in increase of critical loads. P14 sample had the highest critical load. In terms of biological evaluation, P14 exhibited the highest cell proliferation and lowest bacterial adhesion. The highest stability and coherence of P14 coating was also confirmed by mechanical test results. Eventually, it was observed that pretreatment technique has great effect on surface properties. In addition, 14 days immersion period was found to be optimum along the investigated immersion times.