Bacterial colonies are encapsulated in an extra-cellular polymeric matrix to form biofilms in a short time and under many environmental conditions. Biofilms are relevant to public health, medicine and environmental sciences, but also to agriculture and industry. Scanning confocal laser microscopy had been used in conjunction with fluorescent lectin probes to obtain digital images of biofilms. Digital image analysis and classification techniques had been used to assess and quantify structure and spatial variability within biofilms. Reconstitution and computation or at least approximation of the biovolume has represented a major concern in studies to assess spatial variability. Our previous experiments have utilized digital images of biofilms to develop a tool that will ideally determine or at least estimate the volume occupied by bacteria in a certain region of the space. Applications may include the study of biofilm formation and growth, bacterial colonization, and determination of enzymatic activities. The use of GIS techniques takes a step further over various image-processing techniques that already exist, combining it with unique powerful analytical tools. In addition to utilizing this approach for natural systems, and calibrating it using fluorescent microspheres, we will compare the efficiency of this method with other methods in use.