Aim: To evaluate the microleakage of self-etch adhesive used with and without pre-etching step, on human dentin contaminated with two different hemostatic agents. Materials and methods Thirty human premolars were collected and class V cavities with standard dimensions were prepared at the cemento-enamel junction on both buccal and lingual surfaces, with the gingival margin is totally placed in dentin. All specimens were randomly assigned into three groups according to the hemostatic agent used (A); A0: No hemostatic agent applied (control group). A1: Hemostop (Dentsply). A2:ViscoStat® (Ultradent). Each group was furtherly subdivided into two subgroups according to the adhesive protocol used (B); B1: Pre-etching was performed. B2: Pre-etching was not performed. The resultant will be six subgroups, each of them was formed of five teeth with 10 cavities (n=10). Filtek™ Z350 XT nano resin was used to restore all cavities according to the manufacturer's instructions. Teeth were exposed to a thermocycling procedure, thereafter, they were stored in artificial saliva for two years. After immersion in 2% methylene blue dye, Microleakage was measured using USB Digital microscope with a built-in camera connected with an IBM compatible personal computer using a fixed magnification of 40X. Results: A two-way unrelated ANOVA showed significant main effects (p=0.0001) for hemostatic agent, with large effect size η2=86.5%, adhesive protocol with medium effect size η2=68.6%, and the interaction between hemostatic agent and adhesive protocol but with small effect size η2=34.2%.The Duncan post-hoc test revealed a significant difference between all groups except between the groups of Viscosat with pre-etching adhesive protocol (0.54 ± 0.03) on one hand and Hemostop with and without pre-etching adhesive protocol (0.53 ± 0.02) and (0.61 ± 0.04), respectively on the other hand (p > 0.05 ). Conclusions: 1. Contamination with hemostatic agents adversely affected the bonding qualities of self etch adhesives applied with and without pre etching step. 2. Dentin etching prior to self etch adhesives minimized the harmful effect of the hemostatic agents