Cyclic diguanosine monophosphate (c-di-GMP) is the key second messenger regulating bacterial biofilm formation related genes. Several c-di-GMP analogues have demonstrated biofilm inhibition activity. In this study, ribose-phosphate macrocyclic skeleton containing 1'-azido groups was constructed, and CDN analogues were prepared via click chemistry. The biofilm formation inhibition activity of the analogues was evaluated, and compound 17 illustrated better activity than c-di-GMP. This high-throughput strategy could be extended to synthesize cyclic analogues for biological research and immunotherapeutic development.