Arthrobacter sp. Sphe3 and Bacillus sphaericus cells were used for Cu(II) biosorption. The effect of contact time, biosorbent dose, equilibrium pH, temperature and the presence of other ions on the efficiency of the process were extensively studied. Optimum pH value and biomass concentration were determined at 5.0 and 1.0 g/l, whereas contact time was found to be 5 and 10 min for Arthrobacter sp. Sphe3 and Bacillus sphaericus biomass, respectively. Equilibrium data fitted very well to Freundlich model (R ²â€‰= 0.996, n = 2.325, K f = 8.141) using Arthrobacter sp. Sphe3. In the case of B. sphaericus, a Langmuir adsorption model [R ²â€‰= 0.996, Q ₘₐₓ = 51.54 mg-Cu(II)/g] showed to better describe the results. Potentiometric titration and Fourier transform infrared (FTIR) spectroscopy showed that amine, carboxyl and phosphate groups participate in Cu(II)-binding. The calculated thermodynamic parameters indicated the spontaneous and feasible nature of Cu(II) biosorption on both biosorbents. Selectivity of Cu(II) biosorption was examined in binary and multi-ions systems with various anions and cations which are commonly found in municipal and industrial wastewater. A specificity towards Cu(II) was observed in binary mixtures with Cl⁻, CO ₃ ⁻² , NO ₃ ⁻ , SO ₄ ⁻² , PO ₄ ⁻³ , Mg+² and Ca+², and As(V) with the maximum uptake capacity remaining constant even at high competitive ion’s concentrations of 200 mg/l. Desorption studies showed that Cu(II) could be completely desorbed from Cu(II)-loaded Arthrobacter strain Sphe3 and B. sphaericus biomass using 1.0 and 0.8 M HCl, respectively, and both bacterial species could be effectively reused up to five cycles, making their application in wastewater detoxification more attractive.