Toxicological Evaluation and Antimicrobial Activity of a Natural Thymol–Eucalyptol-Based Mixture

Currently, safe alternatives with very low toxicity and good antimicrobial activity are being sought to replace chemical compounds that can be harmful to animal and human health. For this reason, this study evaluated the safety and biofunctional microbiocidal potential of an extract composed of thymol and eucalyptol. Toxicity tests showed low toxicity in both chickens (2000 mg/kg bw) and <i>Artemia salina</i> (EC₅₀ = 2003 mg/L) and <i>Daphnia magna</i> (EC₅₀ = 87 mg/L), indicating a safe usage profile. Oxidative stress biomarkers (nitrite and MDA) and antioxidant enzymes (SOD and catalase) improved in treated chickens at 20 days of age. The hematological and biochemical parameters of the treated birds showed normal values similar to those of the control group chickens, with better protein levels and lower AST levels. Histology of the kidney, intestine, and liver showed no changes in any group, confirming the absence of systemic adverse effects. At the molecular level, an improvement in the expression of tight junction proteins (claudin and occludin) was observed, suggesting a strengthening of the intestinal barrier integrity. Finally, the extract demonstrated an antimicrobial effect (<i>E. coli</i>, <i>C. perfringens</i>, <i>Salmonella</i> sp. and <i>Pseudomonas</i> sp.) comparable to that of organic acids commonly used as food preservatives, positioning it as a promising alternative in applications.