In the present work, sedimentary microbial fuel cells (s-MFC) have been proposed as effective tools to power remote sensors in different aquatic environments, thanks to their ability to produce renewable and sustainable energy continuously and autonomously. The present work proposes the optimization of cylindrical sedimentary microbial fuel cells (s-MFC) as a compact and cost-effective system suitable to be integrated as a payload in an Autonomous Underwater Vehicle (AUV). To this purpose, a new AUV payload, named MFC-payload, is designed to host the cylindrical s-MFC and a data acquisition system to collect and store information on the voltage produced by the cell. Its overall performance was evaluated during two field measurement campaigns carried out in the Mediterranean Sea. This investigation demonstrates the power production by s-MFC during operation of the AUV in seawater and analyzes the actual influence of environmental conditions on the output power. This study demonstrates that energy production by s-MFCs integrated in AUV systems is decoupled by the navigation of the autonomous vehicle itself, showing the effectiveness of the application of MFC-based technology as a power payload for environmental analysis. All these latter results demonstrate and confirm the ability of the devices to continuously produce electricity during different AUV operation modes (i.e., depth and speed), while changing environmental conditions (i.e., pressure, temperature and oxygen content) demonstrate that cylindrical s-MFC devices are robust system that can be successfully used in underwater applications.