The present work aims to elucidate the possibility of injecting ozone into surface waters combined with urban wastewaters in order to improve the water quality of the High Atoyac Sub-basin (HAS) in Central Mexico. For this purpose, twenty physicochemical parameters, eight heavy metals, seven organic compounds, and one biological indicator were assessed in water from different sites of the studied area (the Alseseca River, the Atoyac River and the Valsequillo Reservoir). Results demonstrated that O3 injection led to the decrease of the aromatic fraction of organic molecules since the Spectral Absorption Coefficient at 254 nanometers (SAC254) reduction was found to be 31.7% in the Valsequillo Reservoir water samples. Maximum Chemical Oxygen Demand (COD) removal was observed to be 60.2% from the Alseseca River with a 0.26 mg O3/mg initial COD dose. Among all the phthalates studied in the present work, Di(2-ethylhexyl) phthalate (DEHP) exhibited the highest concentration (5.8 μg/L in the Atoyac River). Treatment with O3 was not effective in eliminating fecal coliforms (FC) in waters that host high organic matter (OM) loads as opposed to waters with low OM. After the injection of 4.7 mg O3/mg COD in the VO3-AT water sample, a 90% removal of Iron (Fe) and Aluminum (Al) was registered; while Manganese (Mn), Nickel (Ni), Zinc (Zn), and Cooper (Cu) showed a 73%, 67%, 81%, and 80% removal, respectively; Chromium (Cr) registered the highest removal (~100%). The present work demonstrated that while finding a suitable O3 dose to improve the quality of water in the HAS, the 5-days Biochemical Oxygen Demand (BOD5)/COD ratio (i.e., biodegradability) is more important than the overall OM removal percentage proving that O3 injection is a feasible process for the treatment of eutrophic waters from HAS.