The aim of this study was to investigate the biocompatibility of nanostructured materials based on highly active calcium silicates mixed with different radiocontrast agents in comparison to MTA+ using in vitro and in vivo model. Morphology of materials' samples was analyzed using SEM while the phase compositions were identified by XRD. pH values of materials' suspensions were conducted by pH-meter. The cytotoxicity of materials' solutions was tested by MTT test (100, 50, 25 and 12.5 mg/ml). LDH and 3H-thymidine assay were utilized for biocompatibility investigations of materials' eluates (24 h, 7 day and 21 day). Eighteen Guinea pigs were used for intramuscular implantation, as teflon tubes with freshly prepared materials were placed into intramuscular pockets. All samples were composed of round and needle-like particles equally distributed with Ca/Si ratio ~2.7 at%, with the presence of hydrated calcium silicate phases. The pH values of ALBO-MPCA1 and ALBO-MPCA2 were high alkaline, while in case of MTA+ they were lower and continuously declined (p < 0.05). Investigated materials didn't exhibit dose-dependent effect on metabolic activity of L929 cells (p > 0.05). Significant differences in the percentage of cytotoxicity between diluted and undiluted extracts between all tested materials after 24 h and 7 day were noticed (p < 0.05). Increase in L929 cells proliferation was noticed in case of undiluted eluates of ALBO-MPCA1 and ALBO-MPCA2 after 7 day (p < 0.05). There were no statistically significant differences in the intensity of inflammatory response between investigated materials and control group after 60 day (p > 0.05). Evaluation of biocompatibility of both ALBO-MPCA1 and ALBO-MPCA2 indicate their potential clinical use.