Therefore, a novel enzymatic treatment with safe level of discharge needs to be developed. Hydrolytic enzymes along with a laccase mediator system have been more beneficial in reducing the pollution load of industries compared to other strategies. An N-hydroxy-based synthetic mediator was predominantly used for this purpose. However, toxicity and cost are two of the major hurdles, which hamper the industrial applications of these synthetic mediators. Therefore, the application of natural mediators in LMS is one of the alternatives to overcome these disadvantages, even though their application may cause grafting onto the pulp, an increase in kappa number, and a reduction in brightness of the pulp. Since chemical-intensive conventional strategies and enzymatic processes utilizing synthetic mediators release high levels of toxic compounds into water bodies, the entire processing of the effluents should be characterized to analyze their ecotoxicity and other hazardous properties. It has been observed that pretreatment with xylanase alone cannot reduce the pollution load of pulp and paper industry significantly. Hence, it is believed that a cocktail of two or more enzymes could reduce the release of hazardous materials to safer levels. Although enzymes are effective at the pretreatment level, effluents from the entire process should be analyzed to study the enzymatic after-effects. Few reports deal with the characterization of effluents from enzyme-aided bleaching processes, but without evaluating the interaction of technical parameters at the pretreatment level. Therefore, in the present investigation, the individual and cumulative effects of physical parameters on the efficiency of both xylanase and laccase were optimized and were analyzed using a statistical model. This is the first ever attempt where ecotoxicity of mixed effluents from the entire bleaching process was characterized using Microtox 81.9% basic toxicity assay method along with the evaluation of the reduction in pollution load in terms of biological oxygen demand and color. A schematic study was done with four different strategies by supplementing the conventional bleaching sequence with xylanase from Bacillus stearothermophilus SDX and Pulpzyme VLBL. In sequential strategies, Ceriporiopsis subvermispora laccase and the commercial laccase TM L603P were used with a natural mediator for the extraction of cellulosic fibers from agro-residual material for paper processing. Owing to the differences in the MK-0683 optimal pH values for the two enzymes and the inhibition of the activity of the mediator system under alkaline conditions, the pulp was subjected to sequential application of xylanase and laccase enzymes. Pretreatment was carried out with xylanase first because the mediators used during laccase pretreatment might generate free radicals capable of hindering the hydrolytic activity of xylanase. Three-dimensional contour plots were also drawn, and it was observed that in xylanase-aided pretreatment, significant decrease in permanganate number was observed.