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Detonation nanodiamonds (NDs) with chlorinated (ND-Cl) and carboxylated (ND-COOH) surfaces were obtained. The broad-spectrum antibiotic Amikacin (Amik) was covalently grafted to the chlorinated surface (ND-Amik) and immobilized by adsorption to carboxylated surface (Amik/ND-COOH). Biological testing in vitro showed the presence of antibacterial activity of the obtained samples against Staphylococcus aureus FDA P209 and Escherichia coli ATCC 25922, close to activity of free amikacin. It was revealed that to maintain antibacterial activity of the samples after their preliminary treatment, important factors such as the use of antioxidants (hydrosulfite and sodium citrate) and lyophilization were necessary.