Extracellular DNA (eDNA) is a major component of bacterial biofilms. In this study, we performed a three-dimensional analysis of biofilm using advanced imaging by confocal laser scanning microscopy (CLSM) and multi-parameter analysis by COMSTAT 2 software, with quantification of and eDNA fluorescence. To investigate the role of eDNA in biofilm, we treated biofilms with DNase I enzyme (DNase), which digested eDNA, and compared DNase treated biofilms and controls. There was a significant reduction of the biomass of biofilms treated with DNase, by spectrophotometry and COMSTAT analysis. The multiparameter analysis evidenced for DNase-treated biofilms a significant decrease in the surface area and the average thickness; opposing to a significant augmentation of the surface/biovolume ratio and the roughness coefficient (Ra*), when compared to controls. We analyzed the parameters of DNase-treated biofilms by Pearson's correlation coefficient and found significant positive correlations between biomass and average thickness; biomass and surface area; surface area and average thickness. On the other hand, there were significant negative correlations between Ra* and biomass; Ra* and average thickness; Ra* and surface area. These findings suggest that eDNA digestion results in biofilm instability and alteration of the three-dimensional architecture, justifying the negative correlation between Ra* and the above-mentioned parameters. In conclusion, our study showed that eDNA digestion produced a massive structural loss, instability, and dramatic changes in the three-dimensional architecture of biofilm. These findings contribute to a better understanding of the role of eDNA and highlight the importance of eDNA as a key component in biofilms.