Flower-like nanomaterials have received great attention due to their large surface area, enhanced charge transfer, and 3D structure, all of which contribute to their applications in drug delivery, cell imaging, biosensors, and other biomedical applications. Two core–shell-structured Si-based Ni composite nanoflowers (Si@NiOOH and Si@Ni) were prepared as NO donors using a modified chemical bath deposition method and thermal reduction. The Si-based Ni composite nanoflowers showed low cytotoxicity toward mouse embryonic fibroblasts when applied at concentrations of up to 2 mg·mL–1. NO-loaded Si@NiOOH and Si@Ni (NO⊂Si@NiOOH and NO⊂Si@Ni, respectively) were obtained by charging Si@NiOOH and Si@Ni with NO at 10 atm for 72 h. The NO storage/release profiles of these materials were then compared via electrochemiluminescence analysis. NO⊂Si@Ni released 0.07 μmol·mg–1 NO over 1.1 h in phosphate-buffered saline at 37 °C, thereby demonstrating that it can fully satisfy the requirements for clinical usage. Furthermore, these nanoflowers presented a composition-dependent NO-delivery ability and excellent antibacterial activities toward Escherichia coli and Staphylococcus aureus. Our study on the NO-delivery capability of bioactive Ni composite nanoflowers can improve the potency of NO donors available for therapeutic applications.