Confocal laser scanning microscopy (CLSM) images were used to quantify in three dimensions the structure of biofilms grown and developed in porous media surfaces. A heterogeneous soil biofilm was observed with a large variety of biological aggregate structures and growth patterns, including cluster-and-protusion type structures, cell aggregate bridging, and a thick bioweb-type growth. An average cell diameter was initially calculated (1.65 µm) using a random stack of optically sliced images; relatively low standard deviation and coefficient of variation were calculated, considering the broad range of micro-organisms present within the biofilm and the presence of varying-size cell clusters. Three volumetric parameters (biovolume, biosurface area, and biothickness) were obtained from 39 biofilm image stacks taken by CLSM in a 15-µm depth. Measured values suggest that there was a large coverage of biofilm over the sand grain surfaces, but due to the heterogeneous structure of the biofilm, considerable variation between images were observed. The high percentage viable biofilm indicated that there was an active and healthy biomass with potential to rapidly reproduce and colonize surfaces. Cell densities were found to be reasonably low for the thin layers of biofilm growing in porous media; areal and volumetric cell densities did not vary much within the biosurface coverage.