Advanced Oxidation Processes and in particular photo-Fenton, represent promising strategies of pathogen inactivation in wastewater effluents. Nevertheless, its full potential is not yet unlocked, as the efficacy of photo-Fenton against viruses has not been deeply explored. In this work, we characterize the effect of major parameters (Fe species and concentration, solar irradiance, pH and microbial competition) on the inactivation of MS2 Coliphage by the photo-Fenton process. The use of Fe(II) salts, under any combination of H2O2 concentration, sunlight irradiance or starting pH (6–8), induced a faster inactivation compared to their Fe(III) counterparts. Moreover, ICP-MS analyses revealed that starting with Fe(II) resulted to higher amount of iron in solution longer than Fe(III), which led to higher inactivation kinetics. Even so, a 4-log MS2 inactivation was achieved upon exposure to 600 W/m2 for 30 min in presence of Fe(III) and H2O2 (1:1 ratio). Furthermore, the inactivation of MS2 was only slightly decreased in presence of the bacterial host, suggesting a low competition for the oxidants in the bulk. The enhancement of iron solubilization through its complexation by organic matter present in wastewater was also investigated, observing an efficient viral inactivation despite the presence of reactive oxygen species (ROS) scavengers. The present data have been used to propose a simple model describing MS2 photo-Fenton inactivation in wastewater. Finally, the pathway describing the photo-Fenton-induced MS2 inactivation in wastewater was proposed, in presence or absence of bacteria.