Acetaminophen (paracetamol, APAP) is one of the fastest growing pharmaceutical pollutants in the environment and has been classified under among the emerging organic pollutants (EOPs). The increasing concentration of it in our environment is not only harmful to the ecosystem, but also to the humans as well. In this study, the microscopy, biochemical test and 16S rRNA sequencing the characterization of APAP as the sole degrading stains viz.Staphylococcus sciuristrain DPP1 (MN744326),Bacillus subtilisstrain DPP3 (MN744327),Bacillus paralicheniformisstrain DKP1 (MN744324),Enterococcus faeciumstrain DKP2 (MN744325) and DDP2 (MT705211) were performed. Haldane's growth kinetic model was used to identify specific growth rate and observed for DPP1 (485 mg/L), DPP3 (593 mg/L), DKP1 (477 mg/L), DKP2 (702 mg/L) and DDP2 (685 mg/L). The maximum specific growth rate was reported for the stains viz. DPP1, DPP3, DKP1, DKP2, and DDP2, was in order of 0.076, 0.223, 0.259, 0.179, and 0.141, respectively. The Box-Behnken Design (BBD) was used to identify the effect of physical parameters on degradation using mathematical modeling. The analysis of variance (ANOVA) showed that the strains DPP1, DPP3, DKP1, DKP2, and DDP2 had significant F-value and regression coefficient (R-2) value of 0.01%, 0.06%, 0.37%, and 0.18%, respectively. The co-culture of the five strains has utilized 1200 mg/L of APAP within 70 h while individual strains took 10 days. The intermediate metabolites like 4-aminophenol, benzamide, (R)-2-methylpentanoic acid, methylene-3-vinyl cyclohexane, and 1,5-hexadiene were identified by GC-MS. The degradation metabolic pathway was predicted by the intermediates by GC-MS, and PathPred based analysis.