Ajwain is a significant medicinal plant known for its antimicrobial, antispasmodic, and digestive properties. Its high resistance to drought allows it to be cultivated in areas with limited water resources. Interestingly, drought stress can increase the production of its active compounds, making the identification of resistant ecotypes crucial for developing sustainable cultivation practices that meet pharmaceutical and industrial demands. This study explored the impact of drought stress on 36 ajwain ecotypes regarding their growth, yield, and phytochemical properties. The findings revealed that under normal conditions, the E-NAJ (80 cm), F-SEP (79 cm), and Y-YAZ (79 cm) ecotypes exhibited the tallest plant heights, while K-RAF (1708 kg ha− 1) and S-B-ZAB (1601 kg ha− 1) demonstrated the highest seed yields (SY). Under drought stress, F-SEP had the best growth at 68 cm, while S-B-ZAB produced the highest yield at 1322 kg ha− 1. In terms of essential oil compounds, the study found that thymol content increased by an average of 28% under stress conditions. The role of monoterpenoid phenols in creating stress tolerance in superior ecotypes such as S-B-ZAB was very prominent. The K-JOO (4.40%) and R-KH-CHE (4.35%) ecotypes yielded the highest essential oil. Cluster analysis classified the ecotypes into four distinct groups, with the third and fourth groups achieving the highest SY (800–816 kg ha− 1) and essential oil production (34–37 kg ha− 1) under stress conditions. An analysis of the synthesis pathway indicated that α-terpinene positively influenced thymol production, while β-phellandrene had a negative effect. Overall, the S-B-ZAB, S-B-ZAH, and K-MAH ecotypes were identified as drought-tolerant varieties with stable both SY and EOY. These findings can inform ajwain breeding programs aimed at cultivation in arid regions. Moreover, the increased essential oil content (EOC) under drought stress presents an opportunity for the targeted production of valuable medicinal metabolites
