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Effective microorganisms pose a great potential in wastewater treatment. In the present study, effective microorganisms’ formulations were developed using different organic substrates that support the growth of more beneficial microorganisms for sewage treatment. Based on the metagenomic analysis and biochemical profile information, the fish waste-based effective microorganisms’ formulation was identified as the effective formulation. Metagenomic analysis showed that fish-based effective microorganisms’ formulation had the Lactobacillus and Acetobacter groups of bacteria. The dominant groups were Lactobacillus pontis (64.85%), Acetobacter aceti (8.92%), and Lactobacillus reuteri (8.98%). The developed fish waste-based effective microorganisms’ formulation was used to treat the sewage water with different concentrations. Effective microorganisms’ formulation at 3% showed appreciable results. It recorded a significant reduction in BOD from 389.2 to 117.9 mg L−1 and COD from 820.5 to 257.1 mg L−1 in 96 h. It also significantly decreased the concentration of ammoniacal and nitrate nitrogen, phosphorus, sulphate, and coliforms. Besides, the effective microorganisms’ formulation ensured the reduced level of odour from sewage water. Therefore, we can effectively use the effective microorganisms' formulation for sewage water treatment and recycling.