The development of reliable models to accurately predict biofilm growth in porous media relies on a good knowledge of the temporal evolution of biofilms structure within the porous network. Since little is known on the true 3D structure of biofilms developed in porous media, this work aimed at developing a new experimental protocol to visualize the 3D microstructure of bacterial biofilms in porous media. The main originality of the proposed procedure lies on the combination of the more recent advances in synchrotron microtomography (Paganin mode) and of a new contrast agent (1-chloronaphtalene) that has never been applied to biofilm visualization. It is shown that the proposed methodology takes advantage of the contrasting properties of 1-chloronaphtalene to prevent some limitations observed with more classical contrast agents. A quantitative analysis of the microstructural properties (volume fractions and specific surface area) of bacterial biofilms developed in columns of clay beads is also proposed on the basis of the obtained 3D images. Biotechnol. Bioeng. 2014;111: 1265–1271. © 2013 Wiley Periodicals, Inc.