This study aimed to investigate the effect of soaking lettuce seeds in a lime oil emulsion at 0 (control), 10, 20, 30, 40, and 50 μl/ml before growing them in a hydroponic plantation. After harvesting baby lettuce at Day 21, all lettuce was kept inside the refrigerator (4 ± 2 C) for the next 7 days. The microbiological quality and the antioxidant properties of lettuce were also investigated. The results revealed that while lime oil emulsion at 10 μl/ml had no effect on seed germination, it delayed the growth of black spot molds and reduced the incidence and severity of black spot fungal disease in baby lettuce by around 87.5%. The morphology of Alternaria sp. and Cercospora sp. shows ruptured conidia and shriveled and damaged mycelium, with a decrease in fungal infection in baby lettuce. In addition, α-terpineol (~53%) was identified from treated baby lettuce after 21 days that was able to inhibit growth of molds. Regarding the microbial eating quality of baby lettuce, mesophiles (~3.0 log 10 CFU/g), psychrophiles (~3.6 log 10 CFU/g), total coliforms (~1.3 log 10 CFU/g), total yeast and mold (~2.0 log 10 CFU/g) and lactic acid bacteria (~3.7 log 10 CFU/g) were significantly reduced in the treated lettuce. The antioxidant capacity in the treated lettuce was higher than the control and maintained stability without affecting its leafy green color and chlorophyll content. This finding is critical for horticulture, particularly in hydroponic vegetable farming. It can be applied in food safety from farm to fork.