The structural components of microorganism are quite related to the toxin and environmental conditions. The vegetative and dormant cells are quite affected by the physical and chemical environments to survive and they will be dormant when they are in the extreme environment. The mechanism to activate the microorganisms however, is not well defined yet in the area of activation state and sporulation state through the analysis of EPS. Other than that even the main mechanism of prior to acquisition of analysis values is not well understood. Therefore, what kind of specific environment to affect the activation and sporulation will be discussed through the analysis of the extracellular polymeric substances(EPS). EPS are a high molecular weight mixture of polymers presenting both outside of cells and interior of microbial aggregates. They are a major complex materials in microbial aggregation for sustaining them together in a three dimensional matrix. Three commonly used extraction methods were applied to this study their effectiveness and quantification in extracting EPS from several Bacillus microorganisms and activated sludge. Three extraction methods used for this study are regular centrifugation with formaldehyde (RCF), Steaming, and EDTA extraction. The results of EPS contents such as the quantitative proteins, carbohydrates and the ratio of protein versus carbohydrate from the several species with the several specific methods showed in this research. This study aims to get comparable results of the quantitative production of EPS and the effectiveness of sedimentation for Bacillus microorganisms and activated sludge from several wastewater treatment plans. The results revealed that the protein amount extracted was the highest by the Steaming method of three extraction methods before sporulation and the carbohydrate amount extracted was the highest by the RCA method of three extraction methods after sporulation. The higher amount of protein compared with carbohydrate from Bacillus microorganisms affected higher sedimentation efficiency, however it could not be found the relation between the EPS production and sedimentation efficiency for the activated sludge.