Breeding new interspecific banana hybrid varieties with pests and disease resistance characters is instrumental for the development of pesticide-free agroecological banana farming systems. Such breeding relies on the combined use of Musa acuminata and M. balbisiana parents. Unfortunately, infectious alleles of endogenous Banana streak virus (eBSV) sequences are present in the genome of M. balbisiana genitors. Upon activation by biotic and abiotic stresses, these infectious eBSVs lead to spontaneous infections by several species of Banana streak virus (BSV) in interspecific hybrids harbouring both M. acuminata and M. balbisiana genomes. In this work, we show that M. balbisiana diploid genitors available for breeding host at least one infectious eBSV. We also show how we segregated infectious and non-infectious eBSV alleles in seedy M. balbisiana diploids through self-pollination or chromosome doubling of haploid lines. We report on the successful breeding of M. balbisiana diploid genitors devoid of all infectious eBSV alleles following self-pollination and on the potential of breeding additional M. balbisiana diploid genitors free of infectious eBSVs by crossing parents displaying complementary eBSV patterns. This work paves the way to the safe use of M. balbisiana genitors for breeding banana interspecific hybrid varieties with no risk of activation of infectious eBSVs.