The ongoing multi-country human monkeypox (HMPX) outbreak was declared as a public health emergency of international concern. Considering the key role of healthcare workers (HCWs) in mitigating the HMPX outbreak, we aimed to assess their level of knowledge and their confidence in diagnosis and management of the disease, besides the assessment of their attitude towards emerging virus infections from a conspiracy point of view. An online survey was distributed among HCWs in Jordan, a Middle Eastern country, during May–July 2022 using a questionnaire published in a previous study among university students in health schools in Jordan. The study sample comprised 606 HCWs, with about two-thirds being either physicians (n = 204, 33.7%) or nurses (n = 190, 31.4%). Four out of the 11 HMPX knowledge items had <50% correct responses with only 33.3% of the study respondents having previous knowledge that vaccination is available to prevent HMPX. A majority of study respondents (n = 356, 58.7%) strongly agreed, agreed or somewhat agreed that the spread of HMPX is related to a role of male homosexuals. Confidence in the ability of diagnosis based on the available monkeypox virus diagnostic tests was reported by 50.2% of the respondents, while the confidence levels were lower for the ability to manage (38.9%) and to diagnose (38.0%) HMPX cases based on their current level of knowledge and skills. Higher confidence levels for HMPX diagnosis and management were found among physicians compared to nurses. The endorsement of conspiracy beliefs about virus emergence was associated with lower HMPX knowledge, the belief in the role of male homosexuals in HMPX spread, and with lower diagnosis and management confidence levels. The current study highlighted the gaps in knowledge regarding HMPX among HCWs in Jordan as well as the lack of confidence to diagnose and manage cases among physicians and nurses. Raising the awareness about the disease is needed urgently considering the rapid escalation in the number of cases worldwide with reported cases in the Middle East. The attitude towards male homosexuals’ role in HMPX spread necessitates proper intervention measures to prevent stigma and discrimination among this risk group. The adoption of conspiratorial beliefs regarding virus emergence was widely prevalent and this issue needs to be addressed with proper and accurate knowledge considering its potential harmful impact.