BACKGROUND AND AIM: The emergence of multidrug-resistant (MDR) Proteus mirabilis in food-producing animals and their associated environments is a growing public health concern. The indiscriminate use of antimicrobials in animal husbandry exacerbates resistance development, posing significant threats to food safety and sustainability. This study investigates the distribution, antibiotic resistance patterns, and virulence-associated genes (VAGs) of P. mirabilis isolated from poultry, livestock, fish, and their environments in Pakistan under a One Health perspective. MATERIALS AND METHODS: A total of 225 samples were collected from poultry (n = 100), livestock (n = 75), and aquatic sources (n = 50) from March 2023 to September 2024. Standard microbiological methods were employed for the isolation and identification of P. mirabilis. Polymerase chain reaction (PCR)-based detection of antibiotic resistance genes and VAGs was performed using specific primers. Antibiotic susceptibility was assessed through the disk diffusion method following Clinical and Laboratory Standards Institute 2022 guidelines. Statistical analyses, including analysis of variance and correlation models, were applied to assess the relationships between variables. RESULTS: P. mirabilis was detected in 28.44% (64/225) of the total samples, with the highest occurrence observed in poultry (38%), followed by livestock (22.67%) and aquatic sources (18%). Resistance to ampicillin (100%), chloramphenicol (82%), cefepime (75%), and ciprofloxacin (75%) was widespread. PCR analysis revealed a high occurrence of extended-spectrum beta-lactamase-producing P. mirabilis carrying bla (CTX-M) (49%), bla (OXA) (54%), and bla (TEM) (25.67%) genes. In addition, VAGs such as zapA (39.53%), ucaA (34.88%), and hpmA (32.55%) were frequently identified. The presence of MDR P. mirabilis in fish and related environments (18%) is alarming, highlighting potential zoonotic and foodborne transmission risks. CONCLUSION: The study underscores the widespread distribution of MDR P. mirabilis in animal-based food sources, raising significant concerns regarding food safety and antimicrobial resistance. The findings reinforce the need for stringent monitoring and regulatory policies to mitigate MDR bacterial dissemination across the food supply chain. Future research should employ metagenomic approaches for comprehensive surveillance and risk assessment.