Background: Limited data is available on Mpox breakthrough infections. Purpose: Investigate a Mpox breakthrough outbreak in three vaccinated individuals. Methods: Study participants provided informed consent. Serology testing was performed in one involved individual (ID-1) using an in-house assay detecting anti-orthopoxvirus IgG. Whole genome sequencing (WGS) was carried out and compared to the reference sequence ON563414.3 (https://www.ncbi.nlm.nih.gov/nuccore/ON563414.3/). Results: Three individuals vaccinated with modified vaccinia Ankara–Bavaria Nordic contracted Mpox following one sexual intercourse event. One of them (ID-1) had received only one vaccine dose, while the other two were fully vaccinated. ID-1 presented to the sexual health clinic of the Universitair Ziekenhuis Brussel with proctitis related to Mpox. Despite one vaccination, serology testing Three months post vaccine showed absence of Mpox virus (MPXV) specific antibodies in ID-1. In contrast, two weeks following the sexual intercourse, seroconversion occurred. WGS of the isolated MPXV showed, compared to the reference sequence, a total of seven single nucleotide variants with four of them indicating protein amino-acid changes. Conclusion: Incomplete MPXV vaccination as well as MPXV variants might result in breakthrough infections. Preventive measures, such as MPVX vaccination, could maintain immunity in individuals with higher risk of MPXV infection, and might lower disease severity.