Background The rapid growth of dairy production in Ethiopia is driven by increasing demand for milk and dairy products. While this transition supports food security and livelihoods, it also increases the risk of infectious diseases and inappropriate use of antimicrobials which can lead to drug residues in milk and contribute to antimicrobial resistance (AMR), posing significant threats to food safety and public health. This study assessed the knowledge, attitudes and practices (KAP) of dairy farming stakeholders regarding veterinary drug residues and AMR in dairy production systems along the Mekelle-Adigrat milk shed, in the Tigray region of Ethiopia.Methods A cross-sectional study was conducted from November 2025 to February 2026 among 528 participants. Data were collected using a structured close-ended questionnaire and analyzed using multivariate logistic regression to identify factors associated with KAP outcomes.Results Overall, 67.2% of respondents had adequate knowledge, 68.2% demonstrated desirable attitudes, and 60.2% reported good practices regarding veterinary drug residues and AMR. Participants’ location, education level, profession, and occupation were significantly associated with KAP scores, while farm size was significantly associated with good practices. Location showed the strongest association with knowledge outcomes. Respondents from Mekelle City had approximately eighteen (AOR:18.4; 95% CI: 10.9–30.7), twenty (AOR:19.5; 95% CI: 11.5–32.9) and five (AOR:4.5; 95% CI: 3.1–6.7) times adequate knowledge, desirable attitude and good practice scores respectively compared with respondents from the Eastern Zone.Conclusion Although most participants demonstrated adequate knowledge and favorable attitudes toward veterinary drug residues and AMR, the relatively lower level of good practices indicates a gap between knowledge and implementation. Strengthening farmer training, veterinary extension services, and regulatory oversight on antimicrobial use is essential to improve responsible antimicrobial use and reduce drug residue risks. These findings provide important evidence to inform targeted One Health-oriented policies and interventions to improve dairy product safety and mitigating AMR in Ethiopia’s rapidly expanding dairy sector.