BACKGROUNDThis study investigates the performance of microbial inoculants by exploring pollutant removal efficiency, microbial viability and substrate removal kinetics for treating polluted surface water. The morphological, elemental composition and biochemical characterization of inoculants were analyzed using field emission scanning electron microscopy with energy dispersive X‐ray spectroscopy (FESEM‐EDX) and Fourier transform infrared (FTIR) techniques. The identification of bacterial strains in polluted feed water also was performed using 16S rRNA partial sequencing. Bench‐scale efficacy of selected microbial inoculants (MI) along with control for each study was performed at variable retention times ranging from 6 to 72 h, under optimum operating conditions.RESULTSThe maximum removal (86% and 90%) of the organic load (chemical and biological oxygen demand, COD and BOD) was achieved by inoculants containing Bacillus subtilis along with other microbial species after 72 h of treatment. The microbial inoculant MI‐2 containing Paracoccus pantotrophus and Pseudomonas Putida exhibited the highest TN removal (66%) after 72 h of contact time. Adenosine triphosphate (ATP) analysis revealed that the active biomass ratio is a more precise indicator of healthy biomass instead of the conventionally used parameter MLVSS (mix liquor volatile suspended solids) in a biological treatment system, which apparently provides a significant addition to the existing knowledge base for the operational wastewater treatment plants (WTPs).CONCLUSIONIn this study, microbial inoculants were found to be frugal and viable treatment tools for the remediation of polluted surface water. Exploiting such inoculants also can overcome the inefficiencies of existing sewage treatment plants by serving as potential tools for bio‐augmenting the indigenous microbial fingerprint of aeration tank/secondary treatment systems. © 2023 Society of Chemical Industry (SCI).