Aim To prepare and characterize Glass ionomer/Collagen hybrid in two different concentrations in comparison with Mineral trioxide aggregate and Glass ionomer in terms of apical leakage, scanning electron microscopic examinations and biocompatibility test. Methodology Sixty extracted teeth were resected, root-end cavities prepared and divided into four equal groups (n=15). Dye extraction method was adopted to evaluate the sealing ability of four equal experimental groups. The different groups were retro-filled with Mineral trioxide aggregate, Glass ionomer, Glass ionomer/Collagen 0.01% hybrid and Glass ionomer/Collagen 1% hybrid. Other samples representing the same groups were investigated using scanning electron microscope. Forty young adult male Wistar Albino rats were used to test each of the four test materials. Each animal received 2 tubes subcutaneously, one tube filled with the experimental material and an empty tube which used as control. Animals were sacrificed after 7 and 60 days from implantation time. The implanted sites were excited and prepared for histological evaluation. Tissue reactions were recorded and scored for the evaluation of biocompatibility. The Data were subjected to Kruskal-Wallis and Mann-Whitney U tests at a significance level of p<0.05. Results Sealing ability results revealed that Glass ionomer/Collagen 0.01% hybrid was as low as Mineral trioxide aggregate while Glass ionomer and Glass ionomer/Collagen 1% hybrid showed relatively high leakage. With respect to biocompatibility results, all materials showed statistically significant decrease in mean inflammatory scores after 60-day observation period, with GI/C hybrid (0.01% and 1%) showing no tissue reaction. Conclusion GI/C hybrid 0.01% can be recommended to be used as retro-grade filling material in comparison to Mineral trioxide aggregate, Glass ionomer and GI/C hybrid 1%.