Additive manufacturing (AM) has the potential to revolutionize the biomaterials field by enabling the affordable and quick production of custom-made implants to fit specific patient needs and anatomy. There already exists literature on the relationships among process parameters, microstructure, and mechanical properties; however, the relationship of complex structures with varied shapes needs further investigation. Therefore, this study sought to determine the effects of AM laser power, exposure time, point spacing, and strut diameter on the microstructure of as-printed Ti-6Al-4V for biomedical application. Mechanical properties of as-printed Ti-6Al-4V samples were assessed by nanoindentation and compared to that of a wrought Ti-6Al-4V control. The AM samples were found to have fine needle-like shape grains, similar to martensite, where the grain size decreased with higher laser power and longer exposure time. All AM samples had low β phase content and the alloying elements were homogenously distributed. Prior β phase colonized with fine α’ phase was identified by the orientation maps—electron backscattered diffraction. Manufacturing defects such as gas porosity and lack of fusion were observed, as well as the presence of cracks. The AM samples were found to have increased hardness and decreased reduced elastic modulus compared to the wrought control. The observed differences in mechanical properties are likely related to the microstructure of these samples. These findings demonstrate that AM alloy microstructure influences both bulk and local properties. This presented study provides additional context into this relationship, furthering the understanding of the complex environment of biomedical implants.