Most mycoplasmas are present as commensals, colonising the mucosa of our respiratory and gastrointestinal tract. Experimental data suggest that the long-term association of certain mycoplasma species with mammalian cells might favour host-cell transformation and malignancy. Moreover, increased mycoplasma infection has been noted in several cancers. Despite efforts to develop target-specific anticancer drugs, current cancer treatment still relies on the use of nucleobase or nucleoside-based analogues. Here, we provide experimental evidence that nucleoside-metabolising catabolic enzymes expressed by mycoplasmas substantially compromise the efficacy of nucleoside antimetabolites used in the treatment of cancer. We also suggest potential methods for improving future chemotherapy by suppressing mycoplasma-mediated catabolism of the anticancer nucleoside analogues.