Strontium-substituted apatites have provoked increased interest in recent years for their beneficial effects on osteoporotic bone treatment and replacement. In this study, rod- and acicular-shaped, strontium-substituted calcium deficient hydroxyapatite (CDHA) nanoparticles with (Ca + Sr)/P ratio of 1.61 were synthesized via accelerated microwave processing. The X-ray powder diffraction analysis indicates the synthesized nanoparticles as apatite phase with diffraction patterns similar to those of hydroxyapatite. The hydrodynamic diameter of the particles were observed to be ~200–500 nm and found to increase with strontium substitution along with an increase in the negative zeta potential by dynamic light scattering method, suggesting the particles to be agglomerates in water. The morphology of the nanoparticles was studied using transmission electron microscopy (TEM), where, pure CDHA showed globular and strontium substituted CDHAs showed rod and acicular shape for 5% and 10% Sr substitution, respectively. The average size of the particles in TEM was measured to be 33 nm × 5 nm, 40 nm × 6 nm, and 55 nm × 8 nm (L × W) for pure and strontium-substituted CDHAs, respectively. Inductively coupled plasma spectroscopy, energy dispersive X-ray analysis, and Fourier transform infrared spectroscopy further confirm the substitution of strontium and deficiency of calcium in the synthesized nanoparticles. Thermal stability and in vitro solubility of CDHA nanoparticles were observed to increase with strontium substitution. The MTT [3-(4, 5-Dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide] assay indicate that the substituted nanoparticles are non-toxic to human periodontal ligament fibroblast (HPDLF) cells. Cell uptake study by fluorescence microscopy using rhodamine-123 and actin/DAPI stained HPDLF cells show cellular localization of the nCDHA, nSr5CDHA, nSr10CDHA nanoparticles without any adverse effects. The strontium-substituted CDHAs showed significant antimicrobial activity against Escherichia coli and Staphylococcus aureus bacteria by colony count method. The 10% Sr substituted CDHA show the maximum microbial reduction of around 56% for E. coli and 35% for S. aureus with 1 × 105 cells/mL of respective bacterial culture.