Abstract Background The persistence of immunological memory after immunization against monkeypox virus (MPXV) is unclear. This study assessed MPXV-specific neutralizing antibodies (NAbs) at >2 years from Mpox or Modified Vaccinia Ankara Bavarian Nordic (MVA-BN) vaccination. Methods Retrospective study including individuals with previous Mpox or vaccination with MVA-BN, whose MPXV-specific NAb titers were already assessed at six months from either event at our center. Neutralizing titers were determined at >2 years from Mpox or vaccination by Plaque Reduction Neutralization Test (PRNT) [positive≥1:10] as the maximum dilution with reduction of cytopathic effect of 90% and 50% (IC90-IC50). Groups were compared using chi-square test, Fisher’s exact test or Wilcoxon rank-sum test. Association between titers at >2 years and individuals characteristics was evaluated with ordinal logistic regression. Results Overall, 48 individuals with previous Mpox and 42 vaccinees were included, all were men. At >2 years from Mpox or vaccination, 22/90 (24.44%) and 53/90 (58.8%) had positive NAb titers respectively at IC90 and IC50. At IC50 previous Mpox was associated with positive titers (68.75% vs 47.62%; p=0.042), with univariate analysis associating higher titers at six months to higher titers at >2 years (OR=6.45,95%CI=1.89–22.07;p=0.003). At multivariate analysis, historical smallpox vaccination was associated to higher titers at >2 years (OR=5.37,95%CI=1.31-22.00;p=0.020), whilst previous Mpox was marginally associated (OR=2.08,95%CI=0.94-4.62;p=0.073). Conclusions MPXV-specific NAbs waned at >2 years from previous infection or vaccination, often becoming undetectable. Higher NAb titers at six months (univariate) and prior smallpox vaccination (multivariate) were associated with NAb detection at >2 years, whilst previous Mpox was marginally associated.