We recently published a new method based on determining cyanobacterial biomass by confocal laser scanning microscopy image analysis (CLSM-IA) (Solé et al., Ultramicrosc 107:669-673, 2007). CLSM-IA allows biomass calculation for microorganisms of a small size, since the limit of the technique's resolution is that generated by a voxel, the smallest unit of a three-dimensional digital image, equivalent to 1.183∈×∈10 -3 mgC/cm3 of sediment. This method is especially suitable for the quantitative analysis of a large number of CLSM images generated from benthic sediments in which complex populations of cyanobacteria are abundant, such as microbial mats. In order to validate the new CLSM approach, mats with varying structural characteristics were studied. We have grouped them into three types: Microcoleus mats (laminated), sandy mats (nonlaminated and composed of well-sorted quartz sands), and oil-polluted mats. In this work, we applied CLSM-IA in natural [the Ebro delta and Sant Jordi colony (Spain), Salins-de-Giraud and Etang de Berre (France), and Orkney Islands (Scotland)] and artificial [mesocosms (Israel)] microbial mats. A total of 4,103 confocal images were obtained in order to determine total and individual cyanobacteria biomass profiles, at microscale level. The data presented in this paper show the efficacy of the method, as it can be applied to highly diverse mat samples. © 2008 Springer Science+Business Media, LLC.