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The restoration of defective bone tissue and complications related to surgery and fracture site infection are major concerns in orthopedic surgeries. However, it is crucial to develop osteoconductive and bacteriostatic composites. Chitosan/nano hydroxyapatite (CT/n-HAp) powder containing of Ag and Si were prepared by an in situ hybridization method. The aim of this work was to elucidate the effect of size, surface roughness, and chemical structure of mentioned nanocomposites on cytotoxicity and bacteriostatic activity via human osteoblast cells and Escherichia Coli, respectively. Particle size, surface roughness, reactive oxygen specious production, and bioactivity of nanocomposites were investigated by X ray diffraction, atomic force microscopy, DPPH assay, and SEM/UV–Visible spectrophotometer, respectively. Bacterial colony counting test, MTT assay and lactate dehydrogenase (LDH) release were performed as bacteriostatic and biocompatibility tests. The results showed that CT/n-HAp/Ag with smaller particle size in the range of 1–22.6 nm (10.00 ± 0.09 nm) than CT/n-HAp/Si in the range of 3–72.5 nm (18.00 ± 0.14 nm) exhibits higher cell viability and bacteriostatic activity, and less LDH release from cell plasma membrane. Integration of Ag into the nanocomposite hindered the release of Ag+ ions and restricts cytotoxic potential on cells. Higher cytotoxic effect of CT/n-HAp/Si might be related to proton concentration derived from nanocomposite and its chemical structure. In conclusion, the strong bone regeneration potential of CT/n-HAp and good biocompatibility and bacteriostatic activity of CT/n-HAp/Ag make it as potential bacteriostatic bone filler in site of infected bone fracture. [ABSTRACT FROM AUTHOR]