Purpose: This study aimed to test bacterial microleakage at the implant-abutment interface (IAI) before and after dynamic loading using a new chewing simulation. Materials and Methods: Fourteen implant systems (n = 5 samples of each) were divided into two groups: (1) systems with conical implant-abutment connections (IACs), and (2) systems with flat IACs. For collecting samples without abutment disconnection, channels (Ø = 0.3 mm) were drilled into implants perpendicularly to their axes, and stainless-steel cannulas were adhesively glued inside these channels to allow a sterilized rinsing solution to enter the implant interior and to exit with potential contaminants for testing. Implants were embedded in epoxy resin matrices, which were supported by titanium cylinders with lateral openings for inward and outward cannulas. Abutments were tightened and then provided with vertically adjustable, threaded titanium balls, which were cemented using composite cement. Specimens were immersed in a bacterial liquid and after a contact time of 15 minutes, the implant interior was rinsed prior to chewing simulation (0 N ⩠ static seal testing). Specimens were exposed to a Frankfurt chewing simulator. Two hundred twenty force cycles per power level (110 in ± X-axis) were applied to simulate a daily masticatory load of 660 chewing cycles (equivalent to 1,200,000 cycles/5 years). The applied load was gradually increased from 0 N to a maximum load of 200 N in 25-N increments. The implant interior was rinsed to obtain samples before each new power level. All samples were tested using fluorescence microscopy; invading microorganisms could be counted and evaluated. Results: No bacterial contamination was detected under static loading conditions in both groups. After loading, bacterial contamination was detected in one sample from one specimen in group 1 and in two samples from two specimens in group 2. Conclusion: Controlled dynamic loading applied in this study simulated a clinical situation and enabled time-dependent analysis regarding the bacterial seal of different implant systems. Conical IACs offer a better bacterial seal compared with flat IACs, which showed increased microleakage after dynamic loading. IAC design plays a crucial role in terms of bacterial colonization. Taking samples of the implant interior without abutment disconnection eliminates an error source. Keywords: abutment disconnection, bacterial microleakage, chewing simulator, implant-abutment connection