We describe the assembly of layer-by-layer (LbL) ultrathin films containing bioactive hydroxyapatite (HAp) rod-shaped nanoparticles with mineralizing capacity. Monodisperse 96 nm long and 9 nm wide HAp nanorods with a surface charge of –14 mV were produced with a microfluidic system. The negatively charged HAp nanorods were assembled with the polycation poly-L-lysine (PLL) in LbL fashion. The successful deposition of alternating layers was confirmed by quartz-crystal microbalance with dissipation monitoring. The Voigt-based viscoelastic model demonstrated steady film growth where three PLL/HAp bilayers reached a thickness of 70 nm. The bioactivity of [PLL/HAp]3 was evaluated in vitro by following the formation of a mineralized hydroxyapatite layer in simulated body fluid (SBF). X-ray diffraction, energy-dispersive X-ray spectroscopy and scanning electron microscopy (SEM) demonstrated formation of a crystalline hydroxyapatite layer and complete surface coverage within 7 days. SaOs-2 osteoblasts-like cells attached to the mineralized surfaces and developed longer filopodia extensions when compared to non-mineralized samples. Our results showed that [PLL/HAp]3 films are feasible osteoconductive coatings applicable to orthopedic implants and fixation devices.