Mitochondrial DNA (mtDNA) depletion syndromes (MDDS) are rare, clinically heterogeneous mitochondrial disorders resulting from nuclear variants in genes of the mitochondrial DNA replication or maintenance machinery. Supplementation with pyrimidine deoxynucleosides have been beneficial in patients and mice with TK2-related MDDS, however, it has not been systematically explored in other forms of MDDS. To investigate the effect of deoxynucleoside supplementation in mitigating the disease in mitochondrial DNA depletion due to pathogenic RRM2B variants, we generated a novel zebrafish knock-out model of this disease and studied the effect of different combinations of deoxynucleosides. Zebrafish larvae carrying a homozygous nonsense mutation in rrm2b present with impaired movement, reduced mtDNA copy number and elevated lactate. Supplementation with different combination of deoxynucleosides was performed, resulting in increased mtDNA copy numbers when supplemented with the two purine deoxynucleosides (dGuo and dAdo), while other combinations had no effect or even further compromised mtDNA copy number in zebrafish. In parallel with increased mtDNA copy number, we detected improved movement and reduction of lactate in the rrm2b-/- fish, confirming the beneficial effect of deoxynucleosides on the whole organism. This treatment did not result in any deleterious effect in wild type and heterozygous fish. Our data suggest that supplementation with deoxynucleosides may be beneficial and should be further investigated in RRM2B-related disease, adding to the growing evidence that it is a valid therapeutic approach which can be trialled for treating a wider range of genetic forms of MDDS.