Implementing strategies for the efficient use of irrigation water is a priority in areas that encounter water shortage. Fennel (Foeniculum vulgare Mill.) is an important medicinal plant grown in many semi-arid regions. A greenhouse study was conducted to investigate the influence of drought stress and the application of three plant growth regulators (PGRs) including jasmonic acid (JA), brassinosteroids (BRs), and putrescine (Put) on photosynthetic parameters, seed yield, essential oil content, essential oil yield, and water use efficiency of seed (WUEseed) of fennel. The results indicated that fennel leaves grown under mild drought stress maintained their relative water content by osmotic adjustment mainly through soluble sugars. Photosynthetic parameters, including leaf chlorophyll index, the maximal quantum yield of PSII photochemistry, net photosynthesis, and transpiration rate were similar in non-stressed plants and mildly-stressed plants prior to flowering. Essential oil content increased in stressed plants, but essential oil yield decreased. WUEseed slightly improved in plants under moderate drought stress. Regardless of the stress level, all three PGRs increased seed yield, primarily by increasing the seed number. Application of BRs, JA, and Put resulted in an approximately 4.2-, 3.5-, and 1.7-fold increase in essential oil yield, respectively. The highest WUEseed (0.82 g/L) was measured in plants that were grown under moderate stress and treated with BRs. Maximum seed yield (2.7 g/plant) was obtained from plants grown under non-stress conditions when treated with JA. It was concluded that spraying fennel plants with PGRs resulted in a considerable increase in essential oil yield.