Surface charge density is a key factor that greatly enhances the performance of a natural-based triboelectric nanogenerator (TENG), which is essential for future sustainable sensing and harvesting devices. This work introduced a conductive interlayer between a main frictional layer and electrode. This approach can suppress the charge recombination rate and improve the amount of charges produced during the triboelectrification process. Bacterial cellulose (BC) film was selected as a main frictional layer for the TENG. A conductive nanomaterial, i.e. silver flake, was incorporated into the BC film as an intermediate layer for enhancing TENG performance. As firstly reported, the maximum electrical outputs for the multi-layer BC structure could be found when using silver flake/BC composite (ratio 1:5) as an intermediate layer, which has 122 V and 8.2 µA of output voltage and current, respectively. This is higher than the output voltage and current of a single layer BC TENG by approximately 3 and 8 times, respectively. The maximum output power of ∼440 µW is achieved by connecting with a load resistor of ∼10 MΩ. This demonstrates an efficient strategy for designing a high performance energy harvester by adding an intermediate layer for the target of practical purposes in sustainable systems.