This study aimed to explore knowledge, attitude, perceptions, and willingness regarding vaccination among university students in Pakistan. This cross-sectional study was carried out using an open online self-administered survey via Google Forms. The survey data were collected between the 15 to 30 of October 2022. A total of 946 respondents participated in the study, of which the majority were female (514, 54.3%). Most students belonged to a medical background, specifically pharmaceutical sciences. Most of the respondents did not know about monkeypox before 2022 (646, 68.3%). Regarding overall knowledge of monkeypox, most of the respondents had average knowledge (726, 76.7%), with very few having good knowledge (60, 6.3%). Regarding overall attitudes towards monkeypox, most of the respondents had neutral attitudes (648, 68.5%). There was a significant association between knowledge of Monkeypox with the type of academic degree (p < 0.001), type of discipline (p < 0.001), and region of respondents (p < 0.001). The willingness to vaccinate among the population was (67.7%). The current study pointed out that the overall knowledge of monkeypox was average in most respondents, with considerable knowledge gaps in most aspects. The overall attitude towards monkeypox was neutral. Further, the knowledge about monkeypox was strongly associated with academic degree, study discipline, and region of respondents. Our findings emphasize the need to raise public awareness by educating students on the monkeypox virus. This will improve adherence to preventative recommendations.