Managing infections caused by multidrug-resistant Gram-negative bacilli is a major public health concern, particularly in hospitals where surfaces can act as reservoirs for resistant microorganisms. Identifying these bacteria in hospital environments is crucial for improving healthcare safety. This study aimed to analyse environmental samples from a veterinary hospital to identify prevalent microorganisms and detect antimicrobial resistance patterns. A total of 183 surface samples were collected from 26 areas at the Veterinary Clinical Hospital of Alfonso X el Sabio University in Madrid. The isolated strains were identified, and susceptibility profiles were determined via the disk diffusion method. Clonality analysis was performed using pulsed-field gel electrophoresis. In total, 109 strains were isolated: 76.15% from the Enterobacteriaceae family and 23.85% non-fermenting Gram-negative bacilli. The isolates included Klebsiella, Enterobacter, Escherichia and Pseudomonas species, which could include high-risk clones, given their ability to carry several antimicrobial resistance genes. The equine area had the highest number of isolates (n = 71), accounting for 65% of the total. High resistance indices were observed against at least five of the 16 antibiotics tested, indicating significant multidrug resistance. Clonality analysis suggested potential cross-transmission within the facility. This study sampled hospital surfaces but not personnel or animals, making contamination sources unclear. Without resampling, the effectiveness of cleaning protocols remains uncertain. Results suggest that hospital staff play a key role in bacterial transmission. The lack of specialised preventive measures in veterinary hospitals highlights a need for further research and improvement.