Mycoplasmas are host-restricted prokaryotes with a nearly minimal genome. To overcome their metabolic limitations, these wall-less bacteria establish intimate interactions with epithelial cells at mucosal surfaces. The alarming rate of antimicrobial resistance among pathogenic species is of particular concern in the medical and veterinary fields. Taking advantage of the reduced mycoplasma genome, random transposon mutagenesis was combined with high-throughput screening in order to identify key determinants of mycoplasma survival in the host-cell environment and potential targets for drug development. With the use of the ruminant pathogenMycoplasma bovisas a model, three phosphodiesterases of the DHH superfamily were identified as essential for the proliferation of this species under cell culture conditions, while dispensable for axenic growth. Despite a similar domain architecture, recombinant Mbov_0327 and Mbov_0328 products displayed different substrate specificities. While rMbovP328 protein exhibited activity towards cyclic dinucleotides and nanoRNAs, rMbovP327 protein was only able to degrade nanoRNAs. The Mbov_0276 product was identified as a member of the membrane-associated GdpP family of phosphodiesterases that was found to participate in cyclic dinucleotide and nanoRNA degradation, an activity which might therefore be redundant in the genome-reducedM.bovis. Remarkably, all these enzymes were able to convert their substrates into mononucleotides, and medium supplementation with nucleoside monophosphates or nucleosides fully restored the capacity of a Mbov_0328/0327 knock-out mutant to grow under cell culture conditions. Since mycoplasmas are unable to synthesize DNA/RNA precursorsde novo, cyclic dinucleotide and nanoRNA degradation are likely contributing to the survival ofM.bovisby securing the recycling of purines and pyrimidines. These results point toward proteins of the DHH superfamily as promising targets for the development of new antimicrobials against multidrug-resistant pathogenic mycoplasma species. Author summary: Mycoplasmas are among the simplest self-replicating organisms. Pathogenic species are of particular concern in the medical and veterinary fields given the alarming rate of antimicrobial resistance documented in these simple, but fast-evolving bacteria. With the use of the ruminant pathogenMycoplasma bovisas a model, several proteins participating in the degradation of cyclic dinucleotides and short RNA molecules were found critical for the survival of this pathogen when grown in the presence of host cells. Remarkably, these essential functions can become dispensable upon the addition of nucleotides into the host-cell culture medium. Since mycoplasmas are unable to synthesize DNA/RNA precursorsde novo, cyclic dinucleotide and nanoRNA degradation are likely contributing to the survival ofM.bovisby securing the recycling of purines and pyrimidines. While illustrating the pivotal role played by nutrients in the mycoplasma host-cell interplay, this study unveils strategies used by reduced-genome bacteria to overcome their limited metabolic capacities and secure their survival in highly sophisticated hosts. These results identified promising targets for the development of new antimicrobials against multidrug-resistant pathogenic mycoplasma species.