Abstract Background Low-value care provides minimal or no benefit for the patient, wastes resources, and can cause harm. Explicit do-not-do recommendations in clinical guidelines are a first step in reducing low-value care. The aim of this study was to identify and prioritize do-not-do recommendations in general practice guidelines with priority for implementation. Methods We used a mixed method design in Dutch primary care. First, we identified do-not-do recommendations through a systematic assessment of 92 Dutch guidelines for general practitioners (GPs), resulting in 385 do-not-do recommendations. Second, we selected 146 recommendations addressing high prevalent conditions. Third, a random sample of 5000 Dutch GPs was invited for an online survey to prioritize recommendations based on the prevalence of the condition and low-value care practice, potential harm, and potential cost reduction on a scale from 1 to 5/6. Total scores could range from 4 to 22. Recommendations with a median score > 12 were included. In total, 440 GPs completed the survey. Results The selection process led to 30 prioritised recommendations. These covered drug treatments (n = 12), diagnostics (n = 10), referral to other healthcare professions (n = 5), and non-drug treatment (n = 3). Conclusion Dutch clinical guidelines include many do-not-do recommendations that are perceived as highly relevant by the GPs. The list of 30 high-priority do-not-do recommendations can be used to raise awareness of low-value care among GPs. As the recommendations are supported with the latest evidence from international studies, primary healthcare professionals and policy makers worldwide can use the list for further validating the list in their local context and designing strategies to reduce low-value care.