Background: Calcium silicate cement like mineral trioxide aggregate (MTA) and Biodentine are known for their biocompatibility and are effectively used as retrograde filling materials. During their placement, the materials interact with oral tissue fluids like saliva and blood, so the aim of the present study was to assess the effect of distilled water, saline, and blood on the surface microstructure and compressive strength of MTA and Biodentine. Materials and methods: In this experimental study, a total of 84 custom-made cylindrical molds were randomly allocated into two main cement groups (n = 42) MTA and Biodentine. Each group was further subdivided into three subgroups (n = 14) as per the testing conditions, that is, samples exposed to distilled water, saline, and fresh blood. Around 10 samples from each subgroup were tested for compressive strength using a universal testing machine (UTM), and the remaining four samples were used to examine the surface characteristics of MTA and Biodentine using a scanning electron microscope (SEM). One-way analysis of variance (ANOVA) and Tukey's post hoc tests were employed to calculate the mean compressive strength and standard deviation values. Results: There was a significant difference in the compressive strength between MTA and Biodentine, especially in the presence of blood. During the SEM analysis, it was found that samples contaminated with blood or saline were devoid of acicular crystals in both groups. MTA group showed a more porous matrix with few hexagonal crystals than Biodentine. Conclusion: Biodentine may be advantageous as a root-end filling or root repair material in the presence of blood.