This study utilized scanning confocal laser microscopy (SCLM) to visualize and analyze microbial biofilms, particularly focusing on *Pseudomonas fluorescens*, *Pseudomonas aeruginosa*, and *Vibrio parahaemolyticus*. SCLM was found to be superior to conventional phase microscopy due to its ability to reject out-of-focus haze and provide higher resolution, especially in thick biofilms with high cell densities. The technique allowed for quantitative computer-enhanced microscopy, enabling detailed analysis of the distribution of cellular and noncellular areas within the biofilm matrices. The results showed that *Pseudomonas* biofilms were more densely packed at their attachment surfaces and became increasingly diffuse near their outer regions, while *Vibrio* biofilms exhibited the opposite trend. The biofilms were generally highly hydrated, open structures composed of 73 to 98% extracellular materials and space. SCLM also revealed the presence of large void spaces within *Vibrio* biofilms and allowed for three-dimensional (3D) reconstructions, which were displayed as stereo pairs. These findings highlight the potential of SCLM in providing detailed insights into the architecture and dynamics of microbial biofilms, which can be crucial for understanding their functional roles in various environments.This study utilized scanning confocal laser microscopy (SCLM) to visualize and analyze microbial biofilms, particularly focusing on *Pseudomonas fluorescens*, *Pseudomonas aeruginosa*, and *Vibrio parahaemolyticus*. SCLM was found to be superior to conventional phase microscopy due to its ability to reject out-of-focus haze and provide higher resolution, especially in thick biofilms with high cell densities. The technique allowed for quantitative computer-enhanced microscopy, enabling detailed analysis of the distribution of cellular and noncellular areas within the biofilm matrices. The results showed that *Pseudomonas* biofilms were more densely packed at their attachment surfaces and became increasingly diffuse near their outer regions, while *Vibrio* biofilms exhibited the opposite trend. The biofilms were generally highly hydrated, open structures composed of 73 to 98% extracellular materials and space. SCLM also revealed the presence of large void spaces within *Vibrio* biofilms and allowed for three-dimensional (3D) reconstructions, which were displayed as stereo pairs. These findings highlight the potential of SCLM in providing detailed insights into the architecture and dynamics of microbial biofilms, which can be crucial for understanding their functional roles in various environments.