This study presents the use of scanning confocal laser microscopy (SCLM) for the optical sectioning of microbial biofilms. SCLM was found to be more effective than conventional phase microscopy for analyzing living biofilms due to its improved rejection of out-of-focus haze and increased resolution. The extent of image improvement varied depending on the characteristics of individual biofilms, being most apparent when films were three-dimensional, thick, or contained a high number of cells. SCLM optical sections were suitable for quantitative computer-enhanced microscopy analyses with minimal interference from overlying or underlying cell material. By combining SCLM with viable negative fluorescence staining techniques, horizontal (xy) and sagittal (xz) sections of intact biofilms of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Vibrio parahaemolyticus were obtained. These sections were analyzed using image-processing techniques to assess the distribution of cellular and noncellular areas within the biofilm matrices. The Pseudomonas biofilms were most cell dense at their attachment surfaces and became increasingly diffuse near their outer regions, whereas the Vibrio biofilms exhibited the opposite trend. Biofilms consisting of different species exhibited distinctive arrangements of the major biofilm structural components. In general, biofilms were found to be highly hydrated, open structures composed of 73 to 98% extracellular materials and space. SCLM allowed for detailed visualization of biofilm structure, including the presence of large void spaces within the Vibrio biofilms. Three-dimensional reconstructions of biofilms were constructed and displayed as stereo pairs. The application of architectural analysis to mixed- or pure-species biofilms allows for detailed examination of the relationships among biofilm structure, adaptation, and response to stress. The study also highlights the advantages of SCLM over traditional microscopy techniques, such as the ability to section biofilms at a fixed point in time and to optically probe fully hydrated biofilms without film disruption. SCLM enables the visualization of biofilm architecture in three dimensions, providing insights into the spatial arrangement of cells, extracellular materials, and spaces. The results demonstrate the potential for noninvasive imaging of intact biofilms and confirm the effective increase in resolution and depth of field through the application of SCLM. The study also shows that SCLM can be used for quantitative imaging, mapping, and display of a broad range of biofilm parameters. The findings suggest that SCLM is a valuable tool for studying microbial biofilms, particularly in conjunction with other light and electron microscopy techniques.This study presents the use of scanning confocal laser microscopy (SCLM) for the optical sectioning of microbial biofilms. SCLM was found to be more effective than conventional phase microscopy for analyzing living biofilms due to its improved rejection of out-of-focus haze and increased resolution. The extent of image improvement varied depending on the characteristics of individual biofilms, being most apparent when films were three-dimensional, thick, or contained a high number of cells. SCLM optical sections were suitable for quantitative computer-enhanced microscopy analyses with minimal interference from overlying or underlying cell material. By combining SCLM with viable negative fluorescence staining techniques, horizontal (xy) and sagittal (xz) sections of intact biofilms of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Vibrio parahaemolyticus were obtained. These sections were analyzed using image-processing techniques to assess the distribution of cellular and noncellular areas within the biofilm matrices. The Pseudomonas biofilms were most cell dense at their attachment surfaces and became increasingly diffuse near their outer regions, whereas the Vibrio biofilms exhibited the opposite trend. Biofilms consisting of different species exhibited distinctive arrangements of the major biofilm structural components. In general, biofilms were found to be highly hydrated, open structures composed of 73 to 98% extracellular materials and space. SCLM allowed for detailed visualization of biofilm structure, including the presence of large void spaces within the Vibrio biofilms. Three-dimensional reconstructions of biofilms were constructed and displayed as stereo pairs. The application of architectural analysis to mixed- or pure-species biofilms allows for detailed examination of the relationships among biofilm structure, adaptation, and response to stress. The study also highlights the advantages of SCLM over traditional microscopy techniques, such as the ability to section biofilms at a fixed point in time and to optically probe fully hydrated biofilms without film disruption. SCLM enables the visualization of biofilm architecture in three dimensions, providing insights into the spatial arrangement of cells, extracellular materials, and spaces. The results demonstrate the potential for noninvasive imaging of intact biofilms and confirm the effective increase in resolution and depth of field through the application of SCLM. The study also shows that SCLM can be used for quantitative imaging, mapping, and display of a broad range of biofilm parameters. The findings suggest that SCLM is a valuable tool for studying microbial biofilms, particularly in conjunction with other light and electron microscopy techniques.