This study presents the synthesis and characterization of two-dimensional (2D) zinc oxide (ZnO) nanosheets (NSs) using a hydrothermal method, followed by their application as an efficient photocatalyst for the degradation of ciprofloxacin (CIP), an antibiotic, under sunlight. The ZnO NSs were also evaluated for their antimicrobial and anticancer activities. The 2D ZnO NSs were found to degrade approximately 90% of CIP within two hours of sunlight exposure. The degradation mechanism was proposed based on ex-situ IR analysis. The ZnO NSs exhibited antimicrobial activity against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria, with minimum inhibitory concentrations (MICs) of 5 μg/l and 10 μg/l, respectively. Additionally, the ZnO NSs showed cytotoxicity against two cancer cell lines, A549 and A375, indicating their potential for biomedical applications. Despite their biocompatibility, the ZnO NSs demonstrated cytotoxic effects on cancer cells, suggesting their potential as a non-toxic alternative in biomedical applications. The ZnO NSs were also found to be effective in degrading antibiotics and other organic contaminants in water, highlighting their potential for environmental remediation. The study concludes that the synthesized ZnO NSs have promising applications in photocatalytic degradation, antimicrobial activity, and anticancer therapy.This study presents the synthesis and characterization of two-dimensional (2D) zinc oxide (ZnO) nanosheets (NSs) using a hydrothermal method, followed by their application as an efficient photocatalyst for the degradation of ciprofloxacin (CIP), an antibiotic, under sunlight. The ZnO NSs were also evaluated for their antimicrobial and anticancer activities. The 2D ZnO NSs were found to degrade approximately 90% of CIP within two hours of sunlight exposure. The degradation mechanism was proposed based on ex-situ IR analysis. The ZnO NSs exhibited antimicrobial activity against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria, with minimum inhibitory concentrations (MICs) of 5 μg/l and 10 μg/l, respectively. Additionally, the ZnO NSs showed cytotoxicity against two cancer cell lines, A549 and A375, indicating their potential for biomedical applications. Despite their biocompatibility, the ZnO NSs demonstrated cytotoxic effects on cancer cells, suggesting their potential as a non-toxic alternative in biomedical applications. The ZnO NSs were also found to be effective in degrading antibiotics and other organic contaminants in water, highlighting their potential for environmental remediation. The study concludes that the synthesized ZnO NSs have promising applications in photocatalytic degradation, antimicrobial activity, and anticancer therapy.