Intermittent slow sand filters for household use have become an established technology that is efficient, simple, and affordable for water treatment. However, bio-clogging can seriously limit hydraulic efficiency and water quality in this treatment system. Therefore, the use of new techniques for studying clogging in slow sand filters has been proposed. This study aimed to evaluate biological clogging using an enzymatic approach, considering also hydraulic aspects and water quality. Six filtration columns with two different granulometric characteristics (fine sand and medium sand) were operated with a downward flow and intermittent flow regime for 120 days. The bio-clogging of the filter media was evaluated considering the presence of esterase enzymes through the degradation of fluorescein diacetate (FDA). So, daily raw and filtered water analyses were performed for physical and chemical characteristics and hydraulic monitoring. The two granulometric characteristics used did not significantly differ between physical and chemical aspects of water quality within the parameters analyzed. The filters showed an average efficiency of turbidity removal of 79.6 % and 80.5 % for fine and medium sand, respectively. The enzymatic assay revealed the existence of microbiological activity throughout the extent of the filters in both granulometric configurations used. The results indicated a potential risk of contamination of the filtered water. The FDA technique proved to be an exciting tool in evaluating biological clogging in intermittent slow sand filters.