The intrinsic characteristics of calcium phosphate-based ceramics (composition, crystallinity, morphology) enable them to be successfully used as osseoconductive coatings on load-bearing implants. Among these compounds, hydroxyapatite (HAP) is the leading material owing to its similarity with the mineral component of bone tissue. In this work, electrostatic spray deposition (ESD) using an organic phosphorus precursor was employed as a suitable one-step coating technique that might overcome the drawbacks of the widely used plasma-spraying process related to extremely high processing temperatures, such as unpredictable phase changes of the feedstock, particle release, and delamination. The key ESD parameters (type of the solvent(s), total reagent concentration, nominal Ca/P ratio in the precursor solution, solution flow-rate, substrate temperature, and nozzleto-substrate distance) were systematically studied to tune, precisely, the microstructure and the crystallite size of single-phase HAP coatings on Ti6Al4V substrates. In vitro studies with MG-63 osteoblast-like cells were performed during 24 h exposure to selected ESD-derived HAP coatings. All coatings were found to support cell viability. Moreover, the coating properties, i.e., crystallite size and microstructure, played an extremely important role in the behavior of osteoblast-like cells. A higher cellular response was observed when low crystalline HAP coatings were employed.