Abstract This research work evaluated the biofilm succession on stone media and compared the biochemical changes of sludge in attached and suspended biological reactors operated under aerobic and anaerobic conditions. Stones incubated (30±2°C) with activated sludge showed a constant increase in biofilm weight up to 5th and 7th weeks time under anaerobic and aerobic conditions respectively, where after reduction (>80%) in MPN index of pathogen indicators on 9th week was recorded. Reduction in parameters like BOD (47.7%), COD (41%), nitrites (60.2%), nitrates (105.5%) and phosphates (58.9%) and increase of DO (176.5%) of sludge were higher in aerobic attached growth reactors as compared to other settings. While, considerable reductions in these values were also observed (BOD, 53.8%; COD, 2.8%; nitrites, 28.6%; nitrates, 31.7% and phosphates, 41.4%) in suspended growth system under anaerobic conditions. However, higher sulphate removal was observed in suspended (40.9 and 54.9%) as compared to biofilm reactors (28.2 and 29.3%). Six weeks biofilm on the stone media showed maximum physiological activities thus, the operational conditions should be controlled to keep the biofilm structure similar to six week old biofilm, and can be employed in fixed biofilm reactors for wastewater treatment.