This study reports the design and preparation of zwitterionic nanocrystalline hydroxyapatite (HA) capable of inhibiting bacterial adhesion while allowing osteoblast cell colonization. The surface functionalization of HA powders was carried out by post-synthesis grafting of 3-aminopropyltriethoxysilane (APTES) and carboxyethylsilanetriol sodium salt (CES) as amine and carboxylate precursors, respectively. The successful functionalization of HA surfaces was assessed by elemental chemical analysis, FTIR, 29Si, 31P and 13C solid state CP/MAS NMR and ζ-potential measurements, and the zwitterionic nature of the synthesized HA was proved through the presence of –NH3+/–COO− pairs on the material surfaces. With the aim of evaluating the feasibility of this functionalization strategy in HA shaped in different physical forms, HA 3D macroporous scaffolds were fabricated by rapid prototyping and then provided with zwitterionic character. The effect of the simultaneous presence of –NH3+/–COO−zwitterionic pairs on the surface of HA on its behaviour regarding bacterial adhesion was tested using E. coli as model bacteria. The in vitro biocompatibility of these materials was investigated with cultured human HOS cells. The results indicate that it is possible to provide HA shaped in different physical forms (particles, granules, coatings, dense blocks, 3D scaffolds, etc.) with bacterial anti-adhesive properties via the “zwitterionization” process without affecting its biocompatibility. These findings open up promising expectations in many clinical fields including dentistry, maxillofacial surgery and otolaryngology, where a decrease in the bacterial adherence onto the implant surface would reduce the infection rates after implantation surgery.