Seed deterioration occurs gradually during long-term seed storage. Viability, vigour, and genetic integrity of African eggplant seeds were assessed under natural ageing during genebank storage. The percentage of tail DNA before and after seed imbibition was used to account for genotoxicity and DNA repair during cold dry storage at 10 °C using the comet assay. After 2, 5, and 12 years of storage in the genebank, a reduction of seed viability was observed in all studied African eggplant genotypes. However, seed viability was more significantly reduced in seed lots that had been stored for a longer period in the genebank (p < 0.05). Overall, higher viability loss was recorded in seeds regenerated 12 years before (p < 0.05). The molecular analysis by comet assay showed significant DNA damage during natural aging under genebank conditions, accompanied by important loss of viability and germination energy in older seeds translated into low DNA repair after seed imbibition. The seed lots which had better initial quality were able to maintain higher viability during genebank storage. The findings inform on the storage behaviour of African eggplant genotypes and are relevant for the proper handling of seed lots in genebanks. They are also relevant for future studies on seed longevity prediction in African eggplant.