Biofilms of Actinobacillus succinogenes have demonstrated exceptional capabilities as biocatalysts for high productivity, titre, and yield production of succinic acid. This work gauges the effectiveness of these biofilms through analysis of the structure and cell viability of biofilms by using confocal scanning laser microscopy, scanning electron microscopy and image analysis software. The structure of the mature six-day-old biofilms of A. succinogenes was characterized by amorphous cell microcolonies of variable area and shape, with a mean thickness of 92 ± 35 μm and low surface area to volume ratios of 0.25 ± 0.07 μm2 μm−3. Biofilm surface area coverage was low near the base (33 ± 13%) but increased towards the outer layer. Despite the presence of water channels at the deeper portions of the biofilm, a greater portion of inactive cells was closest to the attachment surface and 65 ± 2% of the biofilm consisted of dead cells. In this way, a cell viability gradient existed where the outer layer exhibits a greater fraction of active cells compared to the base layer. Biofilm cells underwent a phenotypic change, expressing a filamentous cell morphology different from rod-like morphology of suspended cells. The filamentous morphology permits extensive cell entanglements within a microcolony which may add to the intactness of microcolonies.