Objective: Endoscopy procedures are frequently performed in gastroenterology clinics, and disinfection cost, corrosion and toxicity are important problems in this area. For this purpose, hybrid nanoflowers at various pHs were synthesized with orthophtaldehyde (OPA), which has an important in disinfection in these clinics, and the hybrid nanostructures obtained by reducing the amount of orthophtaldehyde use were aimed to also increase the effectiveness of the disinfectant. Methods: OPA nanoflowers (OPA NFs) were synthesized and their effective diameters (hydrodynamic diameters) and surface charges were determined by dynamic light scattering (DLS) and Zeta potential (ZP) measurements, respectively. Antimicrobial activities of orthophtaldehyde and OPA NFs against Staphylococcus aureus (S. aureus) ATCC 25923, Escherichia coli (E. coli) ATCC 35218 and Candida albicans (C. albicans) ATCC 90028 standard strains were evaluated by the liquid microdilution method using percent inhibition method. Results: OPA-based OPA-Cu2+hybridnano flowers (OPANF) were synthesized successfully at different pH values (pH 7.4, 9 and 11). The most effective antimicrobial activity was observed in the nanoflowers synthesized at pH=7.4 for all tested microorganisms. Although the antimicrobial activity decreased as the pH value increased, NFs activity was higher than OPA alone at all pH values (p<0.001). In this study, NFs synthesized with 0.02 mg/ml OPA were found to be 5.2 times more effective for C.albicans, 5.75 times for E.coli and 4.4 times for S.aureus than OPA(0.02 mg/ml). Conclusion: NFs showed very high antimicrobial activity compared to OPA and also promise to be a preferred agent in medical device disinfection providing a high level of disinfection with the use of a few amounts of OPA that use in clinics.