Enterovirus 70 (EV70) is an emerging viral pathogen that remains viable in final treated effluent. Solar irradiation is, therefore, explored as a low-cost natural disinfection strategy to mitigate potential concerns. EV70 was exposed to simulated sunlight for 24 h at a fluence rate of 28.67 J/cm2/h in three different water matrices, namely, phosphate-buffered saline (PBS), treated wastewater effluent, and chlorinated effluent. In the presence of sunlight, EV70 decreased in infectivity by 1.7 log, 1.0 log, and 1.3 log in PBS, effluent, and chlorinated effluent, respectively. Irradiated EV70 was further introduced to host cell lines and was unable to infect the cell lines. In contrast, EV70 in dark microcosms replicated to titers 13.5, 3.3, and 4.2 times the initial inoculum. The reduction in EV70 infectivity was accompanied by a reduction in viral binding capacity to Vero cells. In addition, genome sequencing analysis revealed five nonsynonymous nucleotide substitutions in irradiated viruses after 10 days of infection in Vero cells, resulting in amino acid substitutions: Lys14Glu in the VP4 protein, Ala201Val in VP2, Gly71Ser in VP3, Glu50Gln in VP1, and Ile47Leu in 3Cpro. Overall, solar irradiation resulted in EV70 inactivation and an inhibition of viral activity in all parameters studied.