In this study, the effects of substrate temperature and ablation wavelength/mechanism on the structural, mechanical, and bioactivity properties of hydroxyapatite (HA) coatings were investigated. HA coatings were deposited on both Si(100) and Ti6Al4V surfaces. Substrate temperature varied from room temperature to 800 °C. Depositions were performed in Ar/H2O and vacuum environments. X-ray diffraction, scanning electron microscopy, and atomic force microscopy techniques were used to analyze structural and morphological variations. The adherence of coatings to the substrates was assessed by the pull-out method. The obtained data indicated that with the temperature increase, the coatings steadily crystallized. However, temperatures above 700 °C adversely affected protein adsorption and adhesion properties. Similar trends were confirmed via pull-out testing, protein adsorption, and cell proliferation tests. The ablation mechanism was also proven to play an important role in the deposition process. Overall, this study provides further evidence that crystallinity is a vital factor in the functionality of the coatings and depends on the deposition conditions. However, all measurements directly indicated that beyond 700 °C, the morpho-structural, mechanical, and bioactivity properties degrade.