Exclusive breastfeeding (EBF) in the first 6 months of life is the most effective way to ensure a child’s survival and optimal development. Despite its benefits, the rates in many countries including Nigeria remain below global targets. This study assessed the knowledge and practice of EBF and identified factors associated with its practice among mothers in Yenagoa, Bayelsa State, Nigeria. A multi-facility cross-sectional study was conducted among 280 mothers of children aged 6–24 months at three healthcare facilities in Yenagoa. Data were collected over three months using a face to face administered structured questionnaire. Statistical analysis was performed to identify factors associated with EBF practice. Results obtained showed that EBF awareness was high, with 94.6% of respondents having heard of it, and 97.1% correctly defining it. Only 21.7% of mothers practiced EBF for the recommended six months. Key barriers included returning to work (38.2%) and perceived milk inadequacy (31.9%). Statistically significant factors positively associated with EBF practice included breastfeeding on demand (p=0.006), maternal conviction of breast milk adequacy (p=0.0007), work-related support for EBF (p=0.003), and family support for EBF (p=0.017). Conversely, mothers who perceived EBF as exhausting/inconveniencing were significantly less likely to exclusively breastfeed (p=0.0001), as well as those who could afford infant formula (p=0.001). This calls for strategies to address the identified huge EBF knowledge –practice gap through strategies that enhance workplace support for EBF, targeted breastfeeding health education for both mothers and available family members and addressing wrong perceptions that impact negatively on EBF.