This study examined the use of various types of activated sludge substrates in greywater treatment. It employed conduit sludge, tofu production waste, and slaughterhouse waste, which were rich in organic compounds, as effective biofiltration agents. These waste materials contained microbial activity capable of degrading pollutants in greywater, making them suitable for pollutant reduction processes. The analysis of waste characteristics in this research aimed to contribute to sustainable wastewater treatment solutions. The objective of this study was to analyze the effect of substrate selection on activated sludge preparation and to assess the impact of activated sludge variations on greywater treatment. The research consisted of two stages. The first stage focused on characterization and cultivation to determine the most viable activated sludge for the subsequent phase. The second stage involved acclimatization and biodegradation, evaluating how the activated sludge adapted to greywater and degraded pollutants. The first stage employed initial characterization followed by cultivation with four treatments: Treatment I, Treatment II, Treatment III, and Treatment IV, with the addition of nutrients. Each sample in each reactor underwent total suspended solids (TSS) analysis at three-day intervals. The best-performing activated sludge was acclimatized in the second stage with greywater under the following conditions: Treatment I contained 300 g of activated sludge and 1,000 mL of greywater; Treatment II contained 300 g of activated sludge and 1,200 mL of greywater; Treatment III contained 300 g of activated sludge and 1,400 mL of greywater, along with a control. At three-day intervals, pH, biochemical oxygen demand (BOD), chemical oxygen demand (COD), TSS, and ammonia levels were analyzed. The results indicated that Treatment IV yielded the highest microbial growth. This fourth treatment was subsequently acclimatized with greywater, and the findings revealed that Treatment III effectively reduced pollutants compared to Treatment I and Treatment II.