The rise in antimicrobial resistance has prompted the development of alternatives to combat bacterial infections. Bald’s eyesalve, a remedy used in the Early Medieval period, has previously been shown to have efficacy against Staphylococcus aureus in in vitro and in vivo models of chronic wounds. However, the safety profile of Bald’s eyesalve has not yet been demonstrated, and this is vital before testing in humans. Here, we determined the safety potential of Bald’s eyesalve using in vitro, ex vivo, and in vivo models representative of skin or eye infections. We also confirmed that Bald’s eyesalve is active against an important eye pathogen, Neisseria gonorrhoeae. Low levels of cytotoxicity were observed in eyesalve-treated cell lines representative of skin and immune cells. Results from a bovine corneal opacity and permeability test demonstrated slight irritation to the cornea that resolved within 10 min. The slug mucosal irritation assay revealed that a low level of mucus was secreted by slugs indicating moderate mucosal irritation. We obtained promising results from mouse wound closure experiments; no visible signs of irritation or inflammation were observed. Our results suggest that Bald’s eyesalve could be tested further on human volunteers to assess safety for topical application against bacterial infections.