Microbial fuel cells (MFCs) in negative pressure environment can provide an increase of 20% in voltage output and a maximum power generation of 7 times compared with that in normal environment. As showed by denaturing gradient gel electrophoresis, the culture in negative pressure environment for 30 days had little effect on diversity of bacterial communities. However, scanning electronic microscope indicated that negative pressure strengthened the attachment between extracellular polymeric substances and carbon felt electrode, it is the main reason for the increase in electrogenic ability of biofilm. The study suggested negative pressure-culture to be a method for promoting the performance of MFCs. The positive effect of negative pressure on the adhesion between biofilm and attachment materials also provides a potential method to improve the performance of biofilm-based environmental technology.