Biofilm plays a significant role in biological hydrogen production. Locomotion and attachment of bacteria are the initial processes in the formation of biofilm. In the present study, bacterial locomotion and attachment in the interspace of packed bed reactor is simulated. The model comprehensively combines flagellar propulsion, Brownian motion, running and tumbling. According to the simulation results, compared with other stacking forms, faster bacterial attachment takes place in the tetrahedron stacking, which can lead to faster biofilm formation. Simulation of bacteria distribution in two-dimensional interspace reveals more bacteria accumulate in the central region of the interspace, while less in the corners. Further study shows that smaller packing or bacteria with smaller cell body in the suspension can lead to faster bacterial attachment in the packed bed reactor. In addition, the suspension viscosity has a tiny effect on the attachment of bacteria. The results can be used as a guideline for the design and operation of packed bed reactors with immobilized biofilm. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.