Essential oils are emerging as alternatives to conventional pest control chemicals. Lippia graveolens Kunth (Mexican oregano) is a source of essential oils and during conventional extraction, the biomass generated is discarded as waste; however, reports show that this biomass is still a rich source of essential oils. Conventional essential oil extraction causes contamination and utilizes toxic solvents. Deep eutectic solvents (DESs) offer low toxicity, biodegradability, high selectivity, and yields comparable to organic solvents. This study obtained essential oil from Lippia graveolens biomass via hydrodistillation with ultrasound-assisted DES pretreatment. This research aimed to optimize the extraction of essential oil from Lippia graveolens biomass using ultrasound-assisted DESs and assess its in vitro and in vivo inhibitory effect on C. asianum. The response variables were extraction yield and total reducing capacity. Optimal conditions were determined using a central composite rotatable design, considering solid-to-liquid ratio (0.38 g/mL), ultrasonic amplitude (45.05%), and time (7.47 min). The optimized oil, with thymol (48%) as the predominant component, exhibited more volatile compounds than conventional hydrodistillation. Fungicidal assays highlighted its potential in controlling anthracnose in papaya fruits caused by C. asianum, making ultrasound-assisted DES pretreatment a promising alternative for obtaining essential oil from botanical byproducts.