The knowledge of pregnant women about Hepatitis B virus infection at three different levels of healthcare and their access to screening and vaccination was evaluated by a questionnaire-based cross-sectional study. There were 643 respondents with a mean age of 30.2 ± 5.2 years and mean gestational age of 26.1 ± 8.4 weeks. The distribution of respondents was 55 (8.6%, primary), 204 (31.7%, secondary) and 383 (59.6%, tertiary) women. The majority of respondents were traders (36%) or civil servants/professionals (28.6%). Overall, 76% of all women had inadequate knowledge about hepatitis B infection; 19.5% had been screened, while 9.7% had been vaccinated. There was an increased likelihood of adequate knowledge, previous screening and vaccination among health workers (p = 0.00). Other positive predictors of knowledge and vaccination were tertiary education (p = 0.04) and tertiary care (p = 0.00). There is inadequate knowledge among pregnant women in Ibadan about Hepatitis B infection, with significant differences at the various levels of care, particularly in non-tertiary settings where screening and vaccination is also sub-optimal. Information dissemination, universal screening and vaccination services for pregnant women in Nigeria require urgent consideration.