Background: Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Streptococcus agalactiae (S. agalactiae) are among the primary pathogens responsible for mastitis on dairy farms. The emergence of antimicrobial resistance (AMR) exacerbates this issue, posing a significant global threat, particularly in veterinary medicine, where mastitis leads to substantial economic losses for the dairy industry. Objectives: This retrospective study aimed to evaluate the resistance patterns of these mastitis-causing pathogens in Iran over a nine-year period (2016–2024). Methods: A total of 5275 milk samples were collected from farms of varying size (small, medium and large). Of these, 960 samples (18.19%) underwent antimicrobial susceptibility testing. Results: Tylosin resistance was the most prevalent among all pathogen isolates, with resistance rates of 46.09% in S. aureus, 61.24% in S. agalactiae, and 91.63% in E. coli. Among the tested pathogens, E. coli exhibited the highest multidrug resistance (MDR) rate at 45.86%, followed by S. aureus (30.4%) and S. agalactiae (22.05%). For S. aureus, resistance to gentamicin increased significantly over time, whereas resistance to cloxacillin showed a marked decline. In E. coli, resistance to penicillin and amoxicillin demonstrated significant upward trends. For S. agalactiae, enrofloxacin resistance declined significantly, while resistance to trimethoprim/sulfamethoxazole increased notably. Farm size was a significant predictor of MDR status, with larger farms associated with lower odds of MDR. Additionally, clinical status was linked to higher MDR odds. Seasonal variations also influenced MDR, with certain seasons showing reduced odds. Conclusions: This study highlighted key factors contributing to MDR in mastitis pathogens. The findings emphasised the complex nature of antimicrobial resistance and the necessity for tailored strategies—such as improved farm management, seasonal interventions, and targeted antimicrobial use—to effectively mitigate resistance.

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