This work evaluated the removal of a mixture of antibiotics from urban wastewater, by a combined process consisting of solar photo-Fenton (SPF) followed by granular activated carbon (GAC). The effects of the SPF process were investigated at a toxicological, microbiological and genomic level, using species of plants and aquatic organisms, bacteria, antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). The results demonstrated that SPF could completely degrade antibiotics present in two types of effluent deriving from a conventional activated sludge system (CAS) and a membrane bioreactor (MBR), operated at the optimum oxidant dose ([H2O2](CAS) = 100 mg/L; [H2O2](MBR) = 50 mg/L) and illumination time (t(CAS) = 115 min; t(MBR) = 111 min) at acidic pH (2.82.9). Moreover, total disinfection was achieved by the SPF process, as cultivable bacteria, including ARB, were inactivated after 60 min of treatment, disabling also the bacterial regrowth after 24 h of storage of the treated effluent. Furthermore, SPF was shown to be effective in degrading the cellular DNA of the effluent, which was reduced to the detection limit after 60 min of treatment in both effluents. The abundance of 16S rRNA was found to be preferentially decreased by the SPF in the MBR-treated effluent (8 folds) compared to the CAS-treated effluent (7.4 folds). The abundance of blaOXA, blaCTX-M, qnrS, sul1, and tetM genes was decreased to the limit of quantification after 60 min of SPF treatment in both effluents. However, the SPF treated flow resulted in increased toxicity, probably due to the oxidation of the dissolved effluent organic matter of the wastewater leading to the formation of toxic products. Therefore, SPF-treated samples collected at different time intervals (30, 60, 90, 120, and 180 min) were subjected to adsorption onto GAC (500 mg/L), and the removal of both the toxicity and the residual antibiotics remaining after SPF, was explored. The combined processes (30 min SPF; 15 min GAC) provided almost complete removal of toxicity and elimination of antibiotics, ensuring wastewater decontamination.