Objective: Relevant animal models to study effects of bacterial aggregates on wound healing are lacking. We aimed at establishing an equine wound model with bacterial aggregates to investigate the impact of bacterial inoculation on normal (thorax) and impaired (limb) wound healing. Approach: Wounds were created on three limbs and both thorax sides of six horses. Twelve out of 20 wounds per horse were inoculated with 104Staphylococcus aureus and 105Pseudomonas aeruginosa on day 4. Healing was monitored until day 27 by clinical assessment, including wound scoring, surface pH measurements, and digital photography for area determination. Biopsies were used for bacterial culture and for peptide nucleic acid fluorescence in situ hybridization to detect bacterial aggregates. Results: Inoculated limb wounds healed slower than noninoculated limb wounds from day 10 onward (p < 0.0001). Inoculated and noninoculated thorax wounds healed equally well and faster than limb wounds. The odds ratio of detecting bacterial aggregates in inoculated limb wounds was 7.1 (2.4–21.0, p = 0.0086) compared with noninoculated limb wounds and 36.2 (3.8–348, p = 0.0018) compared with thorax wounds. Innovation: This equine wound model with bacterial aggregates might be superior to other animal wound models, as both normal and impaired healing can be studied simultaneously. In this model, many aspects of wound healing, including novel treatments, may be studied. Conclusions: The impaired healing observed in inoculated limb wounds may be related to the persistent bacterial aggregates. Both in capability of clearing inoculated bacteria from the wounds and in healing pattern, thorax wounds were superior to limb wounds.