Calcium phosphates (CaP) are extensively studied as additives to dental care products for tooth enamel protection against caries. However, it is not clear yet whether substituted CaP could provide better enamel protection. In this study we produced, characterized and tested in vitro substituted and co-substituted calcium deficient hydroxyapatite (CDHAp) with Sr2+ and F− ions. X-ray powder diffractometry, Fourier transformation infrared spectroscopy, scanning electron microscopy, energy-dispersive X-ray analysis, Brunauer–Emmett–Teller were used to characterize synthesized powders and also cytotoxicity was evaluated. pH = f(t) test was performed to estimate, weather synthesized CDHAp suspensions are able to increase pH of experimental media after acid addition. Synthesis products were incorporated into paste to perform in vitro remineralization on the bovine enamel. In addition to mentioned instrumental methods, profilometry was used for evaluation of remineralised enamel samples. The obtained results confirmed formation of CDHAp substituted with 1.5–1.6 wt% of fluoride and 7.4–7.8 wt% of strontium. pH = f(t) experiment pointed out that pH increased by approximately 0.3 within 10 min after acid addition for all CDHAp suspensions. A new layer of the corresponding CDHAp was formed on the enamel. Its thickness increased by 0.8 ± 0.1 μm per day and reached up to 5.8 μm after 7 days. Additionally, octa calcium phosphates were detected on the surface of control samples. In conclusion, we can assume that CDHAp substituted with Sr2+ and/or F− could be used as an effective additive to dental care products promoting formation of protecting layer on the enamel, but there was no significant difference among sample groups.