Cyanobacteria are the dominant primary producers in microbial mats, which are stratified benthic microbial ecosystems found in coastal environments. Some cyanobacteria form long filaments, which make difficult to apply classical methods to estimate their biomass because they establish strong interactions with detritic particles. In a previous study, we described a method for determining cyanobacterial biomass by means of confocal laser scanning microscopy (CLSM). However, the manual method used, based on summa projection images, was difficult to apply when analyzing a large number of samples. In this paper, we described a new automated method, based on stacks and applying the plugin voxel counter in the ImageJ analysis system, more adequate for obtaining biomass data quickly from a large number of CLSM images.