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In the present research work, we report the co-precipitation synthesis of size‐controlled of uncoated (Magn) and natural chalcone dye-functionalized superparamagnetic magnetite nanoparticles (Magn_NCD NPs) and the study of their efficiency for the adsorption of methylene blue (MB) as well as their antibacterial activity. The NCDwas isolated from Acacia cyanophylla andcharacterized by using IR, UV–visibleand 1H NMRspectroscopies. For the functionalized NPs, the size-control was achieved by varying the pH (pH5 for the sample Magn_NCD5and pH8 for the sample Magn_NCD8) of the reaction medium. The powders consisted of almost spherical NPswith an average particle size of ∼8, ∼3 and ∼7 nm, for Magn, Magn_NCD5and Magn_NCD8, respectively. Adsorption study of the MBonto the NPswas conducted by varying the NPs’ nature, the pH, the NPs’ dosage, the MBconcentration and the contact time. Regarding the effect of the NPs’ nature on the removal of MB, the smallest NPs(Magn_NCD5) were noticeably more performant. For these NPs, the removal efficiency was found to increase with the increase of pH in the pH range 3–9. Additionally, the adsorption kinetics was studied by the three most applicable kinetic models. The pseudo-second order kinetic model was better suited to the experimental adsorption data. Moreover, the adsorption isotherms were analyzed using the Langmuir, Freundlich, Sips and Redlich–Peterson models. The experimental data were best fitted to the Sips model with an extremely high correlation coefficient. Based on this model, the maximum adsorption capacity was calculated (qm= 125 mg.g−1). The reusability of Magn_NCD5was also tested. These NPsshowed a good reusability; After 5 regeneration-reuse cycles, the decrease in MBremoval % was less than 8%. Finally, the antimicrobial activity was carried out against P. aeruginosa(gram-negative) and E. faecalis(gram-positive) bacteria. The Magn_NCD5showed more zone of inhibition in E. faecalis(19 mm) than P. aeruginosa(14 mm).