To address the increasing contamination of aquatic environments and incidence of waterborne diseases, advanced oxidation processes with activated persulfate have emerged as tools to inactivate wastewater microorganisms and contaminants. In this work, the disinfection of a secondary effluent from a wastewater treatment plant by iron-based persulfate activation was studied. Experiments in a batch stirred tank reactor were carried out to evaluate the performance along reaction time and the effect of operational parameters in the oxidative process efficiency (oxidant and iron concentration, pH and temperature). After 60 min of reaction, persulfate and iron concentrations of 3 mM and 0.75 mM, respectively, combined with a neutral initial pH (7.5) and a temperature of 40 °C, allowed to reach values below the detection limit (<10 CFU/100 mL) of enterococci and enterobacteria with and without ciprofloxacin resistance, as well as a 91% inactivation of total heterotrophic organisms and a 70% removal of total organic carbon. Regrowth of microorganisms was evaluated 72 h after treatment and it was only noticed a slight increase in total heterotrophs. Evaluation of physico-chemical characteristics of the treated water showed that it meets the requirements imposed by European and Portuguese legislation for its reuse in irrigation and most urban utilities.