Introduction Evidence-based practice (EBP) leads to improved health outcomes and reduces variability in the quality of care. However, literature on the knowledge, attitudes and use of EBP among midwives is scarce internationally and in Belgium. Methods A cross-sectional study using an online semi-structured questionnaire explored practice, attitudes and barriers on EBP and clinical practice guidelines. Midwives (n=251) working in university and non-university hospitals, primary care, and midwifery education, in Flanders (Belgium) were included. Results Midwives with a Master’s degree (57.7% vs 37.8%; p=0.004), ≤15 years since graduation (50.8% vs 35.5%; p=0.015) and aged <40 years (49.7% vs 34.6%; p=0.02), had better knowledge of the EBP-definition. The majority searched for literature (80.1%), mainly evidence-based (EB) clinical practice guidelines (50.6%), randomized controlled trials (45.0%) and systematic reviews (43.0%). Midwives found EBP necessary and realistic to apply in daily practice and support decision-making. They were willing to improve EBP-knowledge and skills but assumed to be competent in providing evidence-based care. Most respondents were convinced of the importance of EB clinical practice guidelines but did not believe guidelines facilitated their practices or enabled them to consider patient preferences adequately. Half of the midwives (55.8%) experienced barriers to EB clinical practice guideline use, mainly lack of time (35.9%), access (19.5%), and support (17.9%). Conclusions Although midwives showed a positive attitude towards EBP, education programs to promote EBP and improve EBP-related knowledge and skills are needed. Future efforts should focus on developing strategies for overcoming barriers and enhancing the consistency of EBP implementation.