Antimicrobial tolerance, the ability to survive exposure to antimicrobials via transient nonspecific means, promotes the development of antimicrobial resistance (AMR). The study of the molecular mechanisms that result in antimicrobial tolerance is therefore essential for the understanding of AMR. In gram-negative bacteria, the second messenger molecule 3’,5’-cyclic adenosine monophosphate (cAMP) has been previously shown to be involved in AMR. In mycobacteria, however, the role of cAMP in antimicrobial tolerance has been difficult to probe due to its particular complexity. In order to address this difficulty, here, through an unbiased biochemical approaches consisting in the fractionation of clear protein lysate from a mycobacterial strain deleted for the known cAMP phosphodiesterase (Rv0805c) combined with mass spectrometry techniques, we identified a novel cyclic nucleotide-degrading phosphodiesterase enzyme (Rv1339) and developed a system to significantly decrease intracellular cAMP levels through plasmid expression of Rv1339 using the constitutive expression system, pVV16. In Mycobacterium smegmatis mc2155, we demonstrate that recombinant expression of Rv1339 reduced cAMP levels 3-fold and resulted in altered gene expression, impaired bioenergetics and a disruption in peptidoglycan biosynthesis leading to decreased tolerance to antimicrobials that target cell wall synthesis such as ethambutol, D-cycloserine and vancomycin. This work increases our understanding of the role of cAMP in mycobacterial antimicrobial tolerance and our observations suggest that nucleotide signaling may represent a new target for the development of antimicrobial therapies.