In response to the emergence of the Monkeypoxvirus (MPXV) in Australia in May of 2022 we developed and evaluated immunoassays for MPXV and Vaccinia Virus (VACV) IgG and IgM antibodies using serum samples from patients with nucleic acid amplification test (NAAT) confirmed mpox and uninfected, unvaccinated controls. Additionally, 47 health care workers (HCWs) receiving two doses of the third-generation smallpox vaccine Modified Vaccinia Ankara – Bavarian Nordic (MVA-BN) undertook serial serum collection to describe the serological response to vaccination. MPXV NAAT positive patients with serum samples collected within the first 14 days after rash onset had detectable IgG and IgM in 9/12 and 5/12 of patients respectively: with maintenance of IgG and disappearance of IgM titers after 60 days. Detectable antibody in subjects historically vaccinated with early generation vaccines against smallpox was found in 7/7 subjects, at a median of 48 years following vaccination. Two doses of the MVA-BN vaccine were necessary for the complete induction of antibodies, with titers peaking in the fourth week following the second dose. The persistence of a detectable response to MVA-BN declined significantly past 6 months following the second dose. Our findings of a robust humoral response following Mpox infection and historic vaccination, with the fleeting detectability of antibody in naïve subjects immunised with two doses of MVA-BN, provide immunological context to recent reports of breakthrough infections of Mpox despite a two-dose regime, and raise questions to the longitudinal antibody response following Mpox infection.