The World Health Organization (WHO) declared the monkeypox outbreak a public health emergency in June 2022. In Pakistan, positive cases of monkeypox were reported in April 2023. Healthcare workers (HCWs) are considered as a front-line force to combat such outbreaks. A questionnaire-based cross-sectional study was conducted among 11 public sector educational institutions in Punjab, Pakistan, during May and June 2023 among final year medical, pharmacy, and nursing students concerning their knowledge of monkeypox. This included the signs/symptoms of monkeypox. Healthcare students were chosen as they are the HCWs of tomorrow. A total of 389 healthcare students participated in the study, with a mean age of 23.17 ± 1.72 years, and the majority were female. The mean knowledge score was 17.69 ± 4.55 (95% CI 17.24–18.14) out of a maximum total knowledge score of 26 (each correct answer was given a score of 1). The proportion of students with good, moderate, and poor knowledge was 21.6%, 43.2%, and 35.2%, respectively. Age (p = 0.017), gender (p < 0.001), and education (p < 0.001) had a significant impact on the knowledge score. In the multivariate linear regression model, education was the only significant factor linked to knowledge scores. Overall, the majority of future HCWs had moderate knowledge of monkeypox. Consequently, educational activities are needed to improve monkeypox-related knowledge among future HCWs. Furthermore, emerging infectious diseases should be routinely incorporated into HCW curricula.