Hydroxyapatite is a promising biomaterial in bone regeneration due to its bioactive properties as well as its structural and chemical similarities to the inorganic phase of bone tissue. Due to lack of antibacterial properties, it is substituted with a silver ion to achieve antibacterial activity. Silver has antibacterial properties, but can be cytotoxic in higher concentrations. To reduce the cytotoxicity of silver ions, it is co-substituted with strontium ion. The aim of this study was to determine the antibacterial activity of samples of hydroxyapatite enriched with silver ions and hydroxyapatite enriched with silver and strontium ions using the disk diffusion method and determining the number of bacteria in suspension. The tested bacteria were Escherichia coli and Staphylococcus aureus as representatives of the most common pathogens in bone infections. Unsubstituted hydroxyapatite did not show antibacterial activity. All hydroxyapatite samples substituted with silver ion and hydroxyapatite samples substituted with silver and strontium ions showed antibacterial activity in both methods, which is evident from the appearance of the inhibition zone and the analysis of the number of living bacteria in the bacterial suspension after contact with the samples for a certain amount of time. The antibacterial activity of hydroxyapatite samples substituted with silver ions was dependent on the contact time and the amount of substituted silver ions.