In this study, as a measure to enhance the antimicrobial activity of biomaterials, the selenium ions have been substituted into hydroxyapatite (HA) at different concentration levels. To balance the potential cytotoxic effects of selenite ions (SeO32−) in HA, strontium (Sr2+) was co-substituted at the same concentration. Selenium and strontium-substituted hydroxyapatites (Se-Sr-HA) at equal molar ratios of x Se/(Se + P) and x Sr/(Sr + Ca) at (x = 0, 0.01, 0.03, 0.05, 0.1, and 0.2) were synthesized via the wet precipitation route and sintered at 900 °C. The effect of the two-ion concentration on morphology, surface charge, composition, antibacterial ability, and cell viability were studied. X-ray diffraction verified the phase purity and confirmed the substitution of selenium and strontium ions. Acellular in vitro bioactivity tests revealed that Se-Sr-HA was highly bioactive compared to pure HA. Se-Sr-HA samples showed excellent antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus carnosus) bacterial strains. In vitro cell–material interaction, using human osteosarcoma cells MG-63 studied by WST-8 assay, showed that Se-HA has a cytotoxic effect; however, the co-substitution of strontium in Se-HA offsets the negative impact of selenium and enhanced the biological properties of HA. Hence, the prepared samples are a suitable choice for antibacterial coatings and bone filler applications.