The properties of activated sludge systems can be characterized using image analysis procedures. When these systems operate with high biomass content, accurate sludge characterization requires samples to be diluted. Selection of the best image acquisition magnification is directly related to the amount of biomass screened. The aim of the present study was to survey the effects of dilution and magnification on the assessment of aggregated and filamentous bacterial content and structure using image analysis procedures. Assessments of biomass content and structure were affected by dilutions. Therefore, the correct operating dilution requires careful consideration. Moreover, the acquisition methodology comprising a 100× magnification allowed data on aggregated and filamentous biomass to be determined and smaller aggregates to be identified and characterized, without affecting the accuracy of lower magnifications regarding biomass representativeness.