The photocalytic degradation of dicloxacillin (DXC) using TiO2 was studied in synthetic and natural waters. The degradation route and the effect of different experimental variables such as pH, applied power, and the initial concentrations of DXC and the catalyst were investigated. The best performances were achieved at a natural pH 5.8 and using 2.0 g L−1 of TiO2 with 150 W of applied power. The photodegradation process followed Langmuir-Hinshelwood kinetics. The water matrix effect was evaluated in terms of degradation efficiency in the presence of organic compounds (oxalic acid, glucose), Fe2+ ion and natural water. An increase in degradation was observed when ferrous ion was part of the solution, but the process was inhibited with all evaluated organic compounds. Similarly, inhibition was observed when natural water was used instead of distilled water. The extent of degradation of the process was evaluated following the evolution of chemical oxygen demand (COD), antimicrobial activity (AA), total organic carbon (TOC) and biochemical oxygen demand (BOD5). Total removal of DXC was achieved after 120 min of treatment and 95% mineralization was observed after 480 min of treatment. Additionally, the total removal of antimicrobial activity and a high level of biodegradability were observed after the photocalytical system had been operating for 240 min.