Studies have reported the resurgence of insect pests that are resistant to chemical and, more recently, to biological pesticides. Diatraea saccharalis is one of the main pests in sugarcane crops and responsible for large losses in the sugar and alcohol industries. The use of plant extracts including garlic essential oil (Allium sativum) which contains allicin, a compound that interferes with biochemical and physiological factors of target insects is considered an efficient and sustainable alternative for integrated pest management. The objective of this study was to test the toxicity of garlic essential oil on D. saccharalis eggs by analyzing rates of unhatched eggs, changes in external morphology, and esterases profile, enzymes related to insect resistance. For this, 24, 48, and 72-h-old eggs were immersed in 0.1, 0.5, and 1% garlic essential oil solutions for 2 min. Observations on embryonic development were made every 24-h for 72-h. Data on unhatched eggs were collected 288-h after treatments. For the esterases profile, eggs at 24-h after treatment with 1% solution were used. Eggs at 24-h-old, showed the least resistance to bioinsecticide, being that, 24-h after treatments with solutions 0.1, 0.5, and 1%, unhatched rates 80, 91.7, and 89.2%, respectively. The absence of embryos, ruptures, and protrusions on the chorionic region, and partial inhibition of EST-9, 10, and 11 were observed 24-h after the bioassays. These results demonstrate that garlic essential oil affects D. saccharalis eggs, partially inhibiting enzymes related to the insect immune system and can be used as a sustainable alternative for the integrated management of this insect pest.