Ti-6Al-4V (wt%) is the most widely used titanium alloy and its additive manufactured (or 3D printed) parts with near net-shape have provided great advantages for biomedical applications. While the impact of surface roughness on the biocompatibility of 3D-printed Ti-6Al-4V part is recognized, further exploration is needed to fully understand this complex relationship. Hence, this study presents a comprehensive evaluation of as-printed Ti-6Al-4V structures, both with and without surface texturing, with particular focus on the fibroblast response. Alongside a flat surface, or as-printed surface, two different types of surface textures: diamond texture and diamond crystal texture, were meticulously designed and printed through laser powder bed fusion (LPBF). The viability, cell adhesion, and morphology of human and murine fibroblasts seeded on the surface patterns was investigated, as well as the distribution of extracellular matrix (ECM) proteins (collagen I, fibronectin). The results demonstrated that the as-fabricated surface morphologies did not impact fibroblast viability, however, a reduced density of human fibroblasts was observed on the diamond texture surface, likely owing to the upright strut structure preventing cell adhesion. Interestingly, spreading of the human, but not murine, fibroblasts was limited by the remaining partially-sintered powders. The relative intensity of ECM protein signals was unaffected, however, ECM protein distribution across the surfaces was also altered. Thus, the as-printed substrates, particularly with diamond crystal struts, present a promising avenue for the cost-effective and efficient fabrication of Ti-6Al-4V components for medical applications in the future.