Human papillomavirus (HPV) is a common sexually transmitted disease worldwide. While burden of HPV-associated cancers and mortality is higher in low-income countries, there is limited data about knowledge of it among health care students and professionals. We assessed awareness and knowledge of HPV, its related diseases, and HPV vaccine among 333 participants, composed of 146 medical students (MSs) and professionals (MPs) and 187 nursing students (NSs) and professionals (NPs) using a 40-question survey between July 2018 and February 2019. Surveys were conducted in English language using both paper and an online version. Most participants reported that they had heard of HPV and cervical cancer. However, 91.76% of MPs and 77.97% of MSs, but only 41.11% of NPs and 36.17% NSs reported knowing that HPV types 16 and 18 caused cervical cancer. Likewise, about two-thirds of MPs and MSs reported having the knowledge that HPV 6 and 11 caused genital warts versus only a little over one-fourth of NPs and NSs. Only 55.91% of NPs and 51.61% of NSs were aware that HPV could cause cancer in both men and women, whereas 42.35% of MPs, 64.41% of MSs, 41.76% of NPs, and 40.66% of NSs were aware that the vaccine could be given to both boys and girls. While medical professionals were relatively more knowledgeable about HPV and related diseases, overall, knowledge about the HPV vaccine was low among all groups. This knowledge gap is concerning and warrants further attention to fight HPV-related public health burden in Nepal.