Human mitochondrial nuclease, EXOG (hEXOG), is essential for repair of damaged mtDNA. It shares sequence homology with the apoptotic endonuclease EndoG. However, apart from being endonuclease, hEXOG also possesses 5′-exonuclease activity which is necessary for the 5′-end damage removal and thus robust DNA repair in human mitochondria. Recently the X-ray crystal structures of hEXOG in apo and dsDNA-bound forms were solved. It was shown that a Wing domain of hEXOG confers a specific 5′-dsDNA exonuclease activity which provides an optimal substrate for subsequent gap-filling and strand ligation steps, thus efficient base-excision repair (BER). Still, the molecular mechanism underlying mitochondrial BER has yet to be clearly defined. Here we report structural and biochemical evidence of 5′-exonuclease activity of hEXOG on ssDNA as well as ssRNA. Our results demonstrate that hEXOG acts as a multifunctional enzyme which may contribute not only to the 5′-end damage removal in mitochondrial BER but also to elimination of unnecessary single-stranded DNA and RNA molecules that may arise during nucleic acid processing in mitochondria.