In this work, the Fenton technology was applied to decolorize methylene blue (MB) and to inactivate Escherichia coli K12, used as recalcitrant compound and bacteria models respectively, in order to provide an approach into single and combinative effects of the main process variables influencing the Fenton technology. First, Box–Behnken design (BBD) was applied to evaluate and optimize the individual and interactive effects of three process parameters, namely Fe2+ concentration (6.0 × 10−4, 8.0 × 10−4 and 1.0 × 10−3 mol/L), molar ratio between H2O2 and Fe2+ (1:1, 2:1 and 3:1) and pH (3.0, 4.0 and 5.0) for Fenton technology. The responses studied in these models were the degree of MB decolorization (D%MB), rate constant of MB decolorization (kappMB) and E. coli K12 inactivation in uLog units (IuLogEC). According to the results of analysis of variances all of the proposed models were adequate with a high regression coefficient (R2 from 0.9911 to 0.9994). BBD results suggest that [H2O2]/[Fe2+] values had a significant effect only on D%MB response, [Fe2+] had a significant effect on all the responses, whereas pH had a significant effect on D%MB and IuLogEC. The optimum conditions obtained from response surface methodology for D%MB ([H2O2]/[Fe2+] = 2.9, [Fe2+] = 1.0 × 10−3 mol/L and pH = 3.2), kappMB ([H2O2]/[Fe2+] = 1.7, [Fe2+] = 1.0 × 10−3 mol/L and PH = 3.7) and IuLogEC ([H2O2]/[Fe2+] = 2.9, [Fe2+] = 7.6 × 10−4 mol/L and pH= 3.2) were in good agreement with the values predicted by the model.