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Background Brucellosis is the most common zoonotic disease in Oman. Studies about genetic diversity of Brucella are limited in the country. This study aimed to genotype Brucella melitensis in human isolates and milk samples using multi-locus variable number tandem repeats analysis (MLVA-14) in Oman. Methods MLVA-14 was employed for forty-nine B. melitensis recovered from human isolates (n = 21), one goat isolate, and milk samples (n = 27). Results Clustering analysis separated the 49 B. melitensis strains into two main clusters including 31 genotypes. In Dhofar Governorate, shared genotypes among different animal species were identified; the same genotypes were found also in human isolates. Moreover, there was a close genetic relationship between human and milk sample strains from Dhofar and AD Dakhiliya Governorates. Phylogeography investigated by Minimum Spanning Tree analysis showed that Omani strains belonged to the East Mediterranean lineage and formed a distinct branch with a close relationship with two strains from the United Arab Emirates. Moreover, eight Omani strains genotyped from milk shared the same MLVA profile as strains from Spain, Portugal, China, India, and Turkey. The caprine isolate was an outlier correlated with a big cluster mostly formed by isolates from China with other strains from Portugal, Kazakhstan, Turkey, Mongolia, Marocco, France and Spain. Conclusions This study highlights the zoonotic nature of B. melitensis transmission from infected livestock to humans and also its circulation among different animal species. The One Health approach is the way to develop policies and programs for disease surveillance and control.