The accumulation of xenobiotic compounds in different environments interrupts the natural ecosystem and induces high toxicity in non-target organisms. Diclofenac is one of the commonly used pharmaceutical drugs that persist in the environment due to its low natural degradation rate and high toxicity. Therefore, this study aimed to isolate potential diclofenac-degrading bacteria, detect the intermediate metabolites formed, and determine the enzyme involved in the degradation process. Four bacterial isolates were selected based on their ability to utilize a high concentration of diclofenac (40 mg/L) as the sole carbon source. The growth conditions for diclofenac degradation were optimized, and bacteria were identified asPseudomonas aeruginosa(S1),Alcaligenes aquatilis(S2),Achromobacter spanius(S11), andAchromobacter piechaudii(S18). The highest percentage of degradation was recorded (97.79 ± 0.84) after six days of incubation forA. spaniusS11, as analyzed by HPLC. To detect and identify biodegradation metabolites, the GC-MS technique was conducted for the most efficient bacterial strains. In all tested isolates, the initial hydroxylation of diclofenac was detected. The cleavage step of the NH bridge between the aromatic rings and the subsequent cleavage of the ring adjacent to or in between the two hydroxyl groups of polyhydroxylated derivatives might be a key step that enables the complete biodegradation of diclofenac byA. piechaudiiS18, as well asP. aeruginosaS1. Additionally, the laccase, peroxidase, and dioxygenase enzyme activities of the twoAchromobacterstrains, as well asP. aeruginosaS1, were tested in the presence and absence of diclofenac. The obtained results from this work are expected to be a useful reference for the development of effective detoxification bioprocesses utilizing bacterial cells as biocatalysts. The complete removal of pharmaceuticals from polluted water will stimulate water reuse, meeting the growing worldwide demand for clean and safe freshwater.