Microcystins are a group of cyanotoxins with known hepatotoxic effects, and their presence in drinking water represents a public health concern all over the world. The main objective of this work was to evaluate the solar photo-Fenton process at near-neutral pH in the degradation of microcystin-LR (MC-LR) under conditions close to those found in bloom episodes, with a high concentration of cell debris and natural organic matter (NOM). The influence of experimental parameters such as Fe2+ and H2O2 concentrations, reaction matrix, and the presence of scavenger ions, as well as ecotoxicity before and after treatment, was also evaluated. The reaction matrix was obtained from Microcystis aeruginosa cultivated in ASM-1 medium (ACE1 and ACE2 extracts). H2O2 and Fe2+ concentrations were optimized by 22 factorial design with the central point in a bench-scale solar reactor, using ACE1 extract, and the improved condition was applied in a compound parabolic collector (CPC) reactor, for the ACE2, natural water (RVW) and natural water with M. aeruginosa crude extract (RVCE). Matrix effect assays indicated that radical scavengers present in the medium were responsible for the decrease in the mineralization rates. The solar photo-Fenton process in the CPC reactor achieved COD (75%) and MC-LR (70%) reduction after 120 min at pH = 7.8, [H2O2]/COD = 3.18 and [H2O2]/[Fe2+] = 10 for the ACE2 sample. When the same conditions were applied to the RVCE sample, the process removed 77% of DOC and up to 99% of MC-LR after 45 min of the reaction. Sinapis alba bioassays showed that there was no increase in ecotoxicity after the solar photo-Fenton treatment. These results demonstrate the potential of the solar photo-Fenton process at neutral pH as an additional step in the treatment of natural matrices contaminated with microcystins. In addition, the work reinforces the importance of bioassays in treatment process monitoring.