Hydroxyapatite (HAp) attracts interest as a biomaterial for use in bone substitution or allografts. In the current work, biomaterial nanocomposites based on HAp and pectin were synthesized by using the double decomposition method, which involved using pectin extracted from fresh cladodes of the prickly pear, Opuntia ficus-indica. The crystallinity, purity, and several analytical techniques like Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy were used to understand the surface’s shape. The results revealed that the produced HAp/pectin nanoparticles are pure, spherical, and amorphous. The spectroscopic data indicated a substantial interaction between HAp and pectin, specifically between Ca (II) and pectin hydroxyl and carboxyl groups. The presence of pectin showed a noticeable influence on the prepared nanocomposite texture and porosity. We further assess the antibacterial and antifungal activity of the developed nanocomposite against a number of pathogenic bacteria and fungi, evaluated by the well diffusion method. In the absence of pectin, the XRD analysis revealed that the HAp nanoparticles had 10.93% crystallinity. When the pectin concentration reached 10 wt.%, it was reduced to approximately 7.29%. All synthesized nanocomposites demonstrated strong antimicrobial activity against both Gram-positive (Staphylococcus aureus and Bacillus cereus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria in addition to various fungi (e.g., Aspergillus fumigatus, Penicillium funiculosum, and Trichoderma viride). This study endorses the HAp/Pectin nanocomposite as an efficient antimicrobial material for biomedical advanced applications.