Two rotavirus (RV) strains (sialidase-resistant Wa and sialidase-sensitive OSU) were irradiated with simulated solar UVA and visible light in sensitizer-free phosphate buffered solution (PBS) (lacking exogenous reactive oxygen species (ROS)) or secondary effluent wastewater (producing ROS). Although light attenuated for up to 15% through the secondary effluent wastewater (SEW), the inactivation efficacies increased by 0.7 log10 for Wa and 2 log10 for OSU compared to those in sensitizer-free phosphate buffered solution (PBS) after 4 h of irradiation. A binding assay using magnetic beads coated with porcine gastric mucin containing receptors for rotaviruses (PGM-MB) was developed to determine if inactivation influenced RV binding to its receptors. The linear correlation between the reduction in infectivity and the reduction in binding after irradiation in sensitizer-free solution suggests that the main mechanism of RV inactivation in the absence of exogenous ROS was due to damage to VP8*, the RV protein that binds to host cell receptors. For a given reduction in infectivity, greater damage in VP8* was observed with sialidase-resistant Wa compared to sialidase-sensitive OSU. The lack of correlation between the reduction in infectivity and the reduction in binding, in SEW, led us to include RNase treatment before the binding step to quantify virions with intact protein capsids and exclude virions that can bind to the receptors but have their capsid permeable after irradiation. This assay showed a linear correlation between the reduction in RV infectivity and RV-receptor interactions, suggesting that RV inactivation in SEW was due to compromised capsid proteins other than the VP8* protein. Thus, rotavirus inactivation by UVA and visible light irradiation depends on both the formation of ROS and the stability of viral proteins.