In this study, a novel polypyrrole/Sodium-Alginate/CuFe₂O₄ magnetic nanostructure (PPy/NA-Alg/CuFe₂O₄) was synthesized by the hydrothermal route and used as an adsorbent in order to eliminate Direct Blue 199 (DB199) dye from wastewater. The characterization of the synthesized PPy/NA-Alg/CuFe₂O₄ was confirmed by investigation of SEM, TEM, FTIR, XRD, and VSM analyses. Five significant operational parameters including initial dye concentration (20–100 mg/L), temperature (25–45 °C), the dose of adsorbent (2–22 mg), contact time (10-210 min), and pH (2-10) were optimized to achieve the greatest removal efficiency of DB199 by a central composite rotatable design (CCRD) combined with response surface methodology (RSM). A high coefficient of determination (R² = 0.99) was calculated by analysis of variance (ANOVA). Under optimal conditions, adsorption dosage = 7.00 mg, temperature = 30.61 °C, initial concentration = 80.00 mg/L, pH = 4.00 and contact time = 159.42 min, the maximum adsorption capacity of the adsorbent (80.36) was highly matched with the predicted response values, illustrating the appropriate of RSM in optimum conditions. The experimental data were matched well to the Langmuir isotherm model and both pseudo-first-order and pseudo-second-order kinetics. Additionally, thermodynamic parameters exhibited spontaneous nature and exothermic reaction. Adsorption–desorption experiments evidenced that the PPy/NA-Alg/CuFe₂O₄ synthesized could be used for up to three successive adsorption cycles. Furthermore, the nanocomposite showed strong antibacterial activity against two model microorganisms, one gram-negative (Escherichia coli) and one gram-positive (Staphylococcus aureus) via disc diffusion and linear cultivation methods.