The study investigates the photocatalytic degradation of antibiotics and the antimicrobial and anticancer activities of two-dimensional (2D) zinc oxide nanosheets (ZnO NSs). 2D ZnO NSs were synthesized using a hydrothermal method and characterized by XRD, SEM, TEM, and XPS. These nanosheets effectively degraded ciprofloxacin (CIP) under sunlight, achieving ~90% degradation within two hours. The molecular mechanism of CIP degradation was proposed based on ex-situ IR analysis. Additionally, the ZnO NSs exhibited antibacterial properties against *Escherichia coli* and *Staphylococcus aureus*, with minimum inhibitory concentrations (MIC) of 5 μg/mL and 10 μg/mL, respectively. Despite their biocompatibility, the ZnO NSs showed cytotoxicity against A549 and A375 cancer cell lines, indicating their potential in biomedical applications. The study highlights the potential of 2D ZnO NSs as a sustainable and environmentally friendly solution for antibiotic degradation and antimicrobial applications.The study investigates the photocatalytic degradation of antibiotics and the antimicrobial and anticancer activities of two-dimensional (2D) zinc oxide nanosheets (ZnO NSs). 2D ZnO NSs were synthesized using a hydrothermal method and characterized by XRD, SEM, TEM, and XPS. These nanosheets effectively degraded ciprofloxacin (CIP) under sunlight, achieving ~90% degradation within two hours. The molecular mechanism of CIP degradation was proposed based on ex-situ IR analysis. Additionally, the ZnO NSs exhibited antibacterial properties against *Escherichia coli* and *Staphylococcus aureus*, with minimum inhibitory concentrations (MIC) of 5 μg/mL and 10 μg/mL, respectively. Despite their biocompatibility, the ZnO NSs showed cytotoxicity against A549 and A375 cancer cell lines, indicating their potential in biomedical applications. The study highlights the potential of 2D ZnO NSs as a sustainable and environmentally friendly solution for antibiotic degradation and antimicrobial applications.