Leukodystrophies are a heterogeneous group of severe genetic neurodegenerative disorders. A multiple mitochondrial dysfunctions syndrome was found in an infant presenting with a progressive leukoencephalopathy. Homozygosity mapping, whole exome sequencing, and functional studies were used to define the underlying molecular defect. Respiratory chain studies in skeletal muscle isolated from the proband revealed a combined deficiency of complexes I and II. In addition, western blotting indicated lack of protein lipoylation. The combination of these findings was suggestive for a defect in the iron-sulfur (Fe/S) protein assembly pathway. SNP array identified loss of heterozygosity in large chromosomal regions, covering the NFU1 and BOLA3, and the IBA57 and ABCB10 candidate genes, in 2p15-p11.2 and 1q31.1-q42.13, respectively. A homozygous c.436C > T (p.Arg146Trp) variant was detected in IBA57 using whole exome sequencing. Complementation studies in a HeLa cell line depleted for IBA57 showed that the mutant protein with the semi-conservative amino acid exchange was unable to restore the biochemical phenotype indicating a loss-of-function mutation of IBA57. In conclusion, defects in the Fe/S protein assembly gene IBA57 can cause autosomal recessive neurodegeneration associated with progressive leukodystrophy and fatal outcome at young age. In the affected patient, the biochemical phenotype was characterized by a defect in the respiratory chain complexes I and II and a decrease in mitochondrial protein lipoylation, both resulting from impaired assembly of Fe/S clusters.