The stability and mechanical properties of hydroxyapatite (HAp)/Chitosan composite materials depend on the dispersion of HAp aggregates in the chitosan matrix and on the chemical interaction between them. Therefore, hexagonal cross-sectioned HAp nanofibers were produced using a microwave-assisted hydrothermal method. Glutamic acid was used to control the HAp crystal growth; thereby, nanofibers were obtained with a preferential crystalline orientation, and they were grown along the “c” axis of HAp crystal structures. This morphology exposed the (300) and (100) crystal planes on the surface, and several phosphate groups and calcium ions were also exposed; they were able to form numerous chemical interactions with the amine, hydroxyl, and carbonyl groups of chitosan. Consequently, the final mechanical resistance of the composite materials was synergistically increased. Nanofibers were mixed with commercial chitosan using a sonotrode to improve their dispersion within the biopolymer matrix and prevent migration. The HAp nanofiber/Chitosan composite materials showed higher mechanical resistance than that observed in similar materials with the same chemical composition that were made of commercial HAp powders, which were used as reference materials. The mechanical resistance under tension of the composite materials made of nanofibers was similar to that reported for cortical bone.