Simple Summary In intensive farming, antimicrobials are heavily used for the prevention and treatment of animal diseases. However, the improper use of antimicrobials has led to the widespread presence of antimicrobial-resistant bacteria and antimicrobial-resistant genes in the environment. In this study, five dairy farms located in Shandong Province were selected, and a total of 223 isolates were collected from various environmental locations within each farm (bedding, sports field, and milking parlor). The most frequently detected bacteria were Fusobacterium and Escherichia. The majority of bacteria displayed resistance to multiple antibiotics. The sulfonamide resistance gene sul1 showed the highest detection rate, which corresponded to the sulfadiazine resistance phenotype. Doxycycline and levofloxacin demonstrated the most effective antibacterial properties. In conclusion, understanding the microbial species present and their antimicrobial resistance profiles aids in focusing efforts toward sustainable antimicrobial use and safeguarding human health.Abstract Antimicrobials are extensively utilized in dairy farms to prevent and control diseases in cattle. However, their use contributes to the emergence of antimicrobial-resistant bacteria (ARB) and antimicrobial-resistant genes (ARG), and these can be transmitted to the environment. Regular monitoring of antimicrobial resistance (AMR) is crucial for implementing effective mitigation strategies. This research aimed to assess the environmental microbial species present on dairy farms in Shandong Province and characterize the antimicrobial resistance profiles of the isolates. Five dairy farms located in Shandong Province were selected, representing the prevalent large-scale farming patterns in the area. Sampling took place from April to June 2022, with a total of 223 isolates collected from various environmental locations within each farm (bedding, sports field, and milking parlor). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was employed to identify the species of the clinical isolates. The main pathogens isolated were Aerococcus viridans (5.38%, n = 12), Corynebacterium xerosis (4.93%, n = 11), and Acinetobacter lwoffii (4.03%, n = 9). Among the bacterial isolates, resistance to lincomycin was highest at 91%, and 88% were resistant to sulfadiazine. Antimicrobial resistance genes were detected in only a small proportion of the isolates, the most common of which was sul1. These findings highlight the necessity for careful evaluation of antimicrobial usage in maintaining their effectiveness in human medicine. Understanding the microbial species present and their antimicrobial resistance profiles aids in focusing efforts toward sustainable antimicrobial use and safeguarding human health.