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Objective: To identify the knowledge and awareness level of human mpox viral infection among healthcare workers in southwestern Nigeria. Methods: A cross-sectional study was conducted in Ekiti State, southwest Nigeria among 316 healthcare workers that were selected through a systematic random sampling. Data were collected with the aid of a semi-structured, self-administered questionnaire. The Chi-square test and binary logistic regression were used to find the association between the independent and dependent variables. The significance level was set at P-value <0.05. Results: Two hundred and twenty-two (70.3%) of the respondents were aged ⩽40 years, mean age (36±9) years, 189 (59.8%) were female, 306 (96.8%) were Christians, and 203 (64.2%) were married. Three hundred and fourteen (99.4%) of the respondents were aware of mpox infection. Main sources of information about mpox were medical education (44.0%), radio/television (32.0%) and newspaper (21.0%). However, among those aware of the disease, 209 (67.0%) demonstrated poor knowledge levels. Longer than 5 years’ experience of medical practice was the only significant predictor of higher knowledge level of the disease (OR 1.76, 95% CI 1.01-3.06; P=0.046). Conclusions: Despite the high awareness level of mpox infection among healthcare workers, there still exists a huge knowledge gap. It is recommended that targeted intervention could be directed towards continuous medical education and simulation exercises on re-emerging infectious diseases like mpox to improve the knowledge of the healthcare workers.