The bacterial second messenger signaling molecule bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) controls important biological processes such as biofilm formation, virulence response, and motility. This second messenger is sensed by macromolecular targets inside the cell, both protein and RNA, which induce specific phenotypic responses critical for bacterial survival. One class of enzymes responsible for regulating the intracellular concentration of c-di-GMP, and therefore the physiological behavior of the cell, consists of the EAL domain phosphodiesterases, which degrade the second messenger to its linear form, pGpG. Here, we investigate how base and backbone modifications of c-di-GMP affect the rate of cyclic dinucleotide degradation by an EAL domain protein (CC3396 from Caulobacter crescentus). The doubly substituted thiophosphate analogue is highly resistant to hydrolysis by this metabolizing enzyme but can still bind c-di-GMP riboswitch targets. We used these findings to develop a novel ribosyl phosphate-modified derivative of c-di-GMP containing 2′-deoxy and methylphosphonate substitutions that is charge neutral and demonstrate that this analogue is also resistant to EAL domain-catalyzed degradation. This suggests a general strategy for designing c-di-GMP derivatives with increased enzymatic stability that also possess desirable properties for development as chemical probes of c-di-GMP signaling.