Eosinophilic oesophagitis (EoE) is a chronic inflammatory disease afflicting the oesophagus and causing lifelong morbidity. Over the last few decades, EoE has significantly increased in prevalence with oral corticosteroids, such as budesonide, being the current mainstay of therapy. Tacrolimus is an immunomodulatory drug with anti-inflammation properties that is not on the conventional therapeutic regimen for EoE but offers a promising alternative non-steroidal treatment for patients who do not respond to diet elimination, proton pump inhibitors (PPIs), or corticosteroids. This study aims to investigate and compare the accumulation between locally delivered budesonide and tacrolimus, using an ex vivo porcine oesophageal model of EoE, over a range of contact times up to 30 min. Budesonide and tacrolimus were solubilised in a cosolvent and surfactant formulation to maintain solvation capacity in artificial saliva. Injured and non-injured (control) porcine oesophageal mucosa were used as surrogates to represent EoE and healthy oesophageal mucosa in humans, respectively, due to the highly similar physiological architecture of the oesophagus. EoE-mimicking oesophageal damage was chemically induced by pancreatic enzymes and bile salts known to dilate intercellular spaces typically observed in EoE pathophysiology where tight junction damage was represented by an irreversible drop in transepithelial electrical resistance (TEER). Whole tissue and basolateral accumulation of budesonide and tacrolimus were quantified after 30 min using liquid chromatography tandem mass spectrometry (LC-MS/MS). Tacrolimus yielded a significant increase (p < 0.05) in injured tissue accumulation (approximately two-fold) in comparison to non-injured tissue, while budesonide yielded no significant difference (p > 0.05) in tissue accumulation between the two. Considering the significant accumulation of tacrolimus in this ex vivo porcine model of EoE, using injury-induced porcine oesophageal mucosa, this study suggests the use of tacrolimus as a targeted local therapy for the treatment of EoE.