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Introduction Limited data exist on the knowledge of and attitudes towards mpox and willingness to accept the repurposed mpox vaccine among vulnerable populations, such as people living with HIV (PLHIV) and men who have sex with men (MSM), in countries with overlapping HIV and mpox epidemics, like Nigeria.Methods A cross-sectional study was conducted from 26 August 2024 to September 2024, involving 290 PLHIV and 14 MSM at two selected health facilities in Rivers State, Nigeria, using systematic and snowball sampling techniques, respectively. A self-administered web-based Google form was used to collect sociodemographic data, mpox knowledge, attitudes towards the disease and willingness to receive the mpox vaccine. The χ 2 test was used to explore associations between sociodemographic factors and vaccine acceptance. Multivariate logistic regression was employed to identify determinants of the willingness to vaccinate.Results Although most participants (72.7%; n=221) were aware of the disease, 54.3% (n=165) had poor knowledge of mpox. While 60.5% (n=184) considered mpox a serious disease, 60.2% (n=182) were willing to receive the mpox vaccine, though concerns about side effects and mistrust in health systems were major barriers. Sex (χ 2=16.19; p<0.001), level of education completed (χ2=37.63; p<0.001) and marital status (χ2=15.01; p<0.019) were associated with knowledge of mpox. Age and knowledge level were significantly associated with willingness to accept the vaccine. Those aged≤24 years (n=14; 100.0%) and those with good knowledge (n=58; 72.5%) of mpox were more willing to accept the vaccine than those with poor knowledge (χ2=7.272; p<0.027).Conclusions The study revealed limited knowledge of mpox, low perceived risk and poor vaccine acceptance, despite relatively high awareness in the study population. Targeted public health education and confidence-building strategies are valuable to boost vaccine uptake in these at-risk groups.