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Abstract In the twenty-first century, water contamination with pharmaceutical residues is becoming a global phenomenon and a threat. Antibiotic residues and antibiotic resistance genes (ARGs) are recognized as new emerging water pollutants because they can negatively affect aquatic ecosystems and human health, thereby posing a complex environmental problem. These nano-adsorbents of the next generation can remove these pollutants at low concentrations. This study focuses on the chemical synthesis of copper oxide nanoparticles (CuONPs) and nano-zero-valent iron (nZVI) used as nano-adsorbents for levofloxacin removal from water samples and antibiotic-resistant genes. The CuONPs and nZVI are initially characterized by transmission electron microscopy, scanning electron microscopy, and X-ray diffraction. The levofloxacin adsorption isotherm on the CuONPS and nZVI shows the best fit with the Langmuir isotherm model, exhibiting correlation coefficients (R 2) of 0.993 and 0.999, respectively. The adsorption activities of CuONPS and nZVI were fitted to a pseudo-second-order kinetic model with correlation coefficients (R 2) of 0.983 and 0.994, respectively. The maximum levofloxacin removal capacity was observed at (89%), (84%), (89%), (88%) and (71.6) at pH 7 and adsorbent dose(0.06 mg/L), initial LEV concentration (1 mg/L), temperature 25 °C, and contact time 120 min for CuONPs. Removal efficiency was (91%), (90.6%), (91%), (89%), and (80%), at pH 7, adsorbent dose(0.06), initial LEV concentration (1 mg/L), temperature 35 °C, and contact time 120 min. The levofloxacin adsorption is an exothermic process for nZVI and CuONPs, according to thermodynamic analysis. A thermodynamic analysis indicated that each adsorption process is spontaneous. Several genera, including clinically pathogenic bacteria (e.g., Acinetobacter_baumannii, Helicobacter_pylori, Escherichia_coli, Pseudomonas_aeruginosa, Clostridium_beijerinckii, Escherichia/Shigella_coli, Helicobacter_cetorum, Lactobacillus_gasseri, Bacillus_cereus, Deinococcus_radiodurans, Rhodobacter_sphaeroides, Propionibacterium_acnes, and Bacteroides_vulgatus) were relatively abundant in hospital wastewater. Furthermore, 37 antibiotic resistance genes (ARGs) were quantified in hospital wastewater. The results demonstrated that 95.01% of nZVI and 91.4% of CuONPs are effective adsorbents for removing antibiotic-resistant bacteria from hospital effluent. The synthesized nZVI and CuONPs have excellent reusability and can be considered cost effective and eco-friendly adsorbents. Graphical Abstract |