Background: While equine patient-mediated introduction of A. baumannii into hospital settings has been documented, its environmental dissemination and the risk of hospital-acquired surgical site infection remain poorly understood. Objective: Therefore, this descriptive observational study examined (a) the environmental distribution of Acinetobacter spp. in an equine university hospital, (b) the impact of the implementation of new hygiene protocols, (c) the specification of resistance patterns, and (d) the evaluation of the presence of Acinetobacter spp. in hospital-acquired wound infections. Methods: During three sampling periods, environmental samples of the stables, the treatment, and surgery areas were collected before and after cleaning and disinfection. After sampling period 1 (December 2021), the cleaning routines were optimized by reviewing the cleaning and disinfection process, as well as including further surfaces in the cleaning schedule for January 2022). This was followed by a second (February 2022) and a third (June 2022) sampling period. During sampling periods 1 and 2, 76 surfaces were sampled; in sampling period 3, only 21 critical surfaces were examined. Samples were cultured on selective agar plates and incubated at 37 °C, with bacterial growth evaluated after 24–48 h. Wound swabs were enriched in broth before culturing. Bacteria were identified using MALDI-TOF mass spectrometry. During the first sampling period, antibiotic susceptibility testing was performed using broth microdilution according to CLSI-vet standards. Results: During each sampling period, Acinetobacter spp. was detected in at least one sample in each of the different areas; however, there was a reduced detection rate from sampling period 1 throughout sampling period 3. The isolates were highly resistant against beta-lactam and macrolide antibiotics but mostly sensitive to fluroquinolones (enrofloxacin, 2.2% resistance; marbofloxacin, 0.0% resistance), aminoglycosides (gentamicin, 6.5% resistance; kanamycin, 8.7% resistance), and tetracyclines. Acinetobacter spp. was not detected in surgical site infections. Conclusions: Environmental persistence of Acinetobacter spp. in an equine clinical setting does not necessarily translate into surgical site infections. Through prudent antibiotic use, the antibiotic susceptibility of the isolates may be perceived.