Midwives play an important role in the implementation of cervical cancer screening. We assessed the knowledge of human papillomavirus (HPV) infection and of its relationship with cervical cancer in 107 midwives and 29 graduating midwifery students. The majority of midwives (78.5%) were aware that a viral infection causes cervical cancer, whereas only 48.3% of the students knew this (p = 0.003). Only one midwife (0.9%) was not aware of HPV infection compared with 10.3% of the students (p = 0.029). Midwives were also more knowledgeable of the relationship between HPV infection and cervical cancer and of the availability of a vaccine against HPV infection (p = 0.005 and p < 0.0001, respectively). In conclusion, Greek midwives have a satisfactory level of knowledge about cervical cancer and HPV infection, in contrast to midwifery students. It is important to better educate midwifery students in order to facilitate the incorporation of HPV testing and vaccination in clinical practice.