In ovo delivery of carvacrol, the primary active compound in oregano essential oil (OEO) has the potential to enhance gut development in broilers. This study aimed to optimize in ovo application of OEO by investigating day and site of injection and delivery of carvacrol to different embryonic tissues. In Experiment 1, 2 d of injection (embryonic day (E) 12 or 17.5) and 3 sites of injection for OEO (air cell, amniotic fluid, or yolk) were evaluated based on hatchability and posthatching performance. Experiment 2 aimed to examine the impact of combining OEO with the nonionic surfactant polysorbate 80 (p80) at ratios to carvacrol of 0:0, 0:1, 0.5:1, and 1:1 on carvacrol concentration in amniotic fluid, blood, and yolk. The concentration of carvacrol was measured at 3, 6, and 9 h after OEO injection either without (0:1) or with (1:1) p80. Injection of OEO on E12 led to a significant lower hatchability compared to E17.5 (P ≤ 0.01; Δ = 9.2%). Injecting OEO into the air cell, amniotic fluid, or yolk at E17.5 did not significantly affect hatchability and posthatching performance. The highest concentrations of carvacrol found in egg tissues were observed when injected together with surfactant at the 1:1 ratio (P ≤ 0.001; 14.45 µM, 16.64 µM, and 124.82 µM, for air cell, amniotic fluid, and yolk, respectively) compared to the 0:0, 0:1 or 0.5:1 ratios. Carvacrol was highest in the amniotic fluid and blood at the first time point (3 h postinjection) and decreased afterward (P ≤ 0.001), whereas the concentration in yolk remained elevated up to 9 h postinjection. In conclusion, the optimization of the in ovo delivery of carvacrol resulted in that early injection (E12) had negative effects on hatchability and should be avoided. The findings also suggest that using a nonionic surfactant was crucial for an effective delivery of carvacrol in ovo and the migration from amniotic fluid to yolk within 3 h. In addition, carvacrol's persistence in yolk may serve as a route for delivery into the gastrointestinal tract via the yolk stalk during the peri-hatching phase, potentially influencing gut development., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)