Hydroxyapatite nanoparticle plays a significant role in the field of biomedical industries such as tissue engineering and regenerative medicine, carriers for drug delivery, photocatalyst, biosensors, and membranes for heavy metal removal from polluted water. The present work aims to synthesize the hydroxyapatite from bio-waste materials like cuttlefish bone using a precipitation method by changing reaction temperatures (room temperature, 60 °C, 70 °C, and 80 °C), and pH (9,10,11, and 12). The structure, particle size, and crystallinity of the obtained HAp were evaluated by XRD analysis. The functional groups present in the HAp nanoparticles were analyzed and confirmed by FTIR spectroscopy. The surface morphology of the particles was analyzed by using FESEM and the particle sizes were ranging from 40 to 160 nm for different pH values. The elemental composition was determined by EDAX analysis. The antibacterial activity of the sample was tested against gram-positive and gram-negative bacteria. The zone of inhibition value against gram-negative bacteria was found to be 20 ± 0.32 mm and 16 ± 0.18 mm against gram-positive bacteria for the sample with a pH value of 10. The obtained results confirmed that the optimized temperature, time, and pH are suitable for the preparation of HAp with excellent desired properties, which is employed as a better candidate for biomedical applications. © 2023 Elsevier B.V., All rights reserved.