Synthetic azo dyes, extensively utilized in textile and allied industries, are a persistent source of environmental contamination due to their chemical stability, xenobiotic nature, and resistance to conventional wastewater treatment. In this study, Lactobacillus casei immobilized on porous clay substrates was evaluated for the biodegradation of six reactive dyes: Reactive Red 198, Reactive Violet 5, Reactive Yellow 3, Reactive Orange 5, Reactive Navy Blue 4, and Reactive Black 5 sourced from textile effluents. Decolorization assays demonstrated efficient removal across concentrations of 250–1000 mg/L, with maximum degradation observed at pH 7, 37 °C, and 72 h under static conditions, achieving removal efficiencies of 90.17% (RR198), 92.24% (RV5), 92.18% (RY3), 94.04% (RO5), 95.66% (RNB4), and 92.18% (RB5). Response Surface Methodology (RSM) using the Box–Behnken design facilitated optimization of operational parameters, and statistical analyses confirmed model adequacy, with predicted and experimental decolorization values in close agreement. By accounting for interactive effects among variables, RSM achieved superior degradation efficiency compared to the single-factor OFAT approach. FTIR analysis revealed the disappearance and shifting of characteristic –N = N– (azo), –OH, and –NH peaks, indicating cleavage and modification of the dye’s functional structure. Complementary HPLC profiling showed the emergence of new peaks with altered retention times, confirming the formation of low-molecular-weight metabolites and providing clear evidence of dye biodegradation. Phytotoxicity assays using Vigna radiata demonstrated that degraded metabolites exhibited minimal toxicity, with plumule and radicle growth comparable to the control. The germination percentage in untreated dye controls was only 20–40%, whereas the degraded dye–treated samples showed significantly higher germination rates of 80–95%. These findings highlight L. casei immobilized on porous clay as a cost-effective, environmentally sustainable, and mechanistically robust strategy for the remediation of azo dye-contaminated wastewater, with potential for scalable industrial application.