Objectives: This study aimed to compare the effect of human blood and platelet-rich fibrin (PRF) on the surface microhardness of hydraulic calcium silicate-based cements (OrthoMTA and RetroMTA). Materials and Methods: Two types of mineral trioxide aggregate, OrthoMTA and RetroMTA, were mixed and placed into cylindrical molds. The lower surfaces of all cements were exposed to saline. The upper surfaces of cements were exposed to human blood, PRF, or phosphate buffer saline (PBS). After storage for 7 days in fully saturated humidity at 37°C, the microhardness of cement surface exposed to blood, PRF, or PBS was measured using the Vickers microhardness test. The data were analyzed using two-way analysis of variance and post hoc Tamhane’s T2 test. The significance level was set at P<0.05. Results: Exposure to blood and PRF significantly decreased the surface microhardness of OrthoMTA and RetroMTA. The microhardness of PBS-contacted cements was significantly higher than that of blood or PRF groups (P<0.001). The microhardness values for OrthoMTA exposed to PRF were significantly higher than the blood group (P=0.020). There were no significant differences between RetroMTA contacted with blood or PRF groups (P=0.985). When exposed to blood or PBS, RetroMTA had a significantly higher microhardness than OrthoMTA (P<0.001 for blood, P=0.002 for PBS). Conclusion: Exposure to blood or PRF decreased the surface microhardness of both cements. Blood-contaminated RetroMTA showed significantly higher surface microhardness than OrthoMTA contacted with blood. No significant difference was found between PRF-contacted OrthoMTA and RetroMTA.