Purpose: The aim of this study was to evaluate the removal torque and in vitro biofilm penetration at Morse taper and hexagonal implant-abutment joints after fatigue tests. Materials and Methods: Sixty dental implants were divided into two groups: (1) Morse taper and (2) external hexagon implant-abutment systems. Fatigue tests on the implant-abutment assemblies were performed at a normal force (FN) of 50 N at 1.2 Hz for 500,000 cycles in growth medium containing human saliva for 72 hours. Removal torque mean values (n = 10) were measured after fatigue tests. Abutments were then immersed in 1% protease solution in order to detach the biofilms for optical density and colony-forming unit (CFU/cm2) analyses. Groups of implant-abutment assemblies (n = 8) were cross-sectioned at 90 degrees relative to the plane of the implant-abutment joints for the microgap measurement by field-emission guns scanning electron microscopy. Results: Mean values of removal torque on abutments were significantly lower for both Morse taper (22.1 ± 0.5 μm) and external hexagon (21.1 ± 0.7 μm) abutments after fatigue tests than those recorded without fatigue tests (respectively, 24 ± 0.5 μm and 24.8 ± 0.6 μm) in biofilm medium for 72 hours (P = .04). Mean values of microgap size for the Morse taper joints were statistically signicantly lower without fatigue tests (1.7 ± 0.4 μm) than those recorded after fatigue tests (3.2 ± 0.8 μm). Also, mean values of microgap size for external hexagon joints free of fatigue were statistically signicantly lower (1.5 ± 0.4 μm) than those recorded after fatigue tests (8.1 ± 1.7 μm) (P .05). The optical density of biofilms and CFU mean values were lower on Morse taper abutments (Abs630nm at 0.06 and 2.9 × 104 CFU/cm2) than that on external hexagon abutments (Abs630nm at 0.08 and 4.5 × 104 CFU/cm2) (P = .01). Conclusion: The mean values of removal torque, microgap size, and biofilm density recorded at Morse taper joints were lower in comparison to those recorded at external hexagon implant-abutment joints after fatigue tests in a simulated oral environment for 72 hours. Keywords: biofilms, dental implants, fatigue, implant-abutment, removal torque