Among Additive Manufacturing (AM) technologies, Laser Powder Bed Fusion (LPBF) has made a great contribution to optimizing the production of customized implant materials. However, the design of the ideal surface topography, capable of exerting the best biological effect without drawbacks, is still a subject of study. The aim of the present study is to topographically and biologically characterize AM-produced Ti6Al4V ELI (Extra Low Interstitial)samples by comparing different surface finishing. Vertically and horizontally samples are realized by LPBF with four surface finishing conditions (as-built, corundum-sandblasted, zirconia-sandblasted, femtosecond laser textured). Bioactivity in vitro tests are performed with human osteoblasts evaluating morphology, metabolic activity, and differentiation capabilities in direct contact with surfaces. Scanning electron microscope and profilometry analysis are used to evaluate surface morphology and samples' roughness with and without cells. All tested surfaces show good biocompatibility. The influence of material surface features is evident in the early evaluation, with the most promising results of morphological study for laser texturing. Deposition orientations seem to influence metabolic activities, with XZ orientation more effective than XY. Current data provide the first positive feedback on the biocompatibility of laser texturing finishing, still poorly described in the literature, and support the future clinical development of devices produced with a combination of LPBF and different finishing treatments.