All health workers who care for women and children during the postnatal period and beyond are key players in protecting, promoting, and supporting breastfeeding. However, most health workers do not have the necessary knowledge and skills to effectively play their roles. This study aims to assess the knowledge about breastfeeding among health workers. This was a cross-sectional study among 220 health workers at the University of Port Harcourt Teaching Hospital. A 32-point questionnaire based on the World Health Organisation (WHO) and the United Nations Children’s Fund (UNICEF) manual on the Baby-Friendly Hospital Initiative Training Course for Maternity Staff was used to assess the knowledge of the health workers. Scores of >50% were classified as good knowledge and <50% as poor knowledge. The overall good knowledge about breastfeeding was 51.8%. The majority (80.0%), had good knowledge about the benefits of breastfeeding and 169 (76.8%) of health workers had good knowledge about exclusive breastfeeding and the timing of breastfeeding. Good knowledge about the importance of colostrum was 27.7%, 91 (41.4%) health workers had good knowledge of birth practices that affect breastfeeding, and only 72 (32%) health workers had good knowledge about the problems of breastfeeding. In conclusion, the overall knowledge about breastfeeding among health workers in this study was not satisfactory. To breach this gap, hospitals should ensure that their staff has sufficient knowledge, competence and skill to support breastfeeding.