The objective was to describe and validate a new and alternative software procedure for 24-locus mycobacterial interspersed repetitive unit-variable number-tandem repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis (Mtb) based on the multipurpose BioNumerics software. DNA from randomly selected isolates of Mtb from two European laboratories, including external control samples for MIRU-VNTR typing, were analysed. Samples were genotyped using the commercial 24-locus VNTR typing kit from GenoScreen. The PCR amplified fragments were separated by capillary electrophoresis. For the subsequent analyses, the currently used software GeneMapper was compared with BioNumerics. The endpoint was the level of concordance when comparing genotyping results obtained from BioNumerics with results obtained from GeneMapper and the ECDC proficiency study reference results. Also, the number of necessary manual standard size corrections and allele assignments in the two different software methods were compared. In total, 272 DNA samples, including the ECDC proficiency panel, were analysed. For all samples, there were 100% concordance of results. For a randomly selected set of 96 samples the numbers of manual corrections needed for size standards were 199 with GeneMapper versus zero for BioNumerics. The numbers of manual corrections for allele assignments were 122 with GeneMapper versus 16 with BioNumerics. In conclusion, we have validated the multipurpose software BioNumerics for standard 24-locus MIRU-VNTR typing and the software shows promising benefits in terms of simplification and minimization of hand-on time.