Fenugreek (Trigonella foenum-graecum L.) is a seed spice grown during winter season in North India mostly on conserve moisture of monsoon rains. An experiment was conducted to investigate the effect of moisture stress on seed yield, total oil, fatty acid composition, phenolic content, and antioxidant activity of 13 fenugreek genotypes with an objective to identify suitable genotypes for water-limiting conditions and judicious application of available irrigation water. Moisture stress was created by withholding irrigation in one set of genotypes at flowering and post-flowering stage. Moisture stress at post-flowering stage adversely affected seed yield of all genotypes. Genotype AFg-6 showed at par seed yield (7.7 g/plant) with non-stress (control) (7.5 g/plant), while genotype AM-327-3 produced more yield when moisture stress was given at flowering stage. Oil content under non-stress environment ranged from a minimum of 2.62% in genotype AFg-6 to a maximum of 5.31 in AM-327-3 which increased up to 3.15 and 5.42% when moisture stress was created at post-flowering stage. GC–MS profiling of seed oil revealed significant genotypic variation in fatty acid composition. Total phenolic, flavonoid, sapogenin, diosgenin content, and antioxidant capacity of methanol crude seed extract increased during moisture stress. Genotypes AFg-4, AFg-6, Hisar Sonali, and RMt-305 have been identified for limited water conditions. Results of analysis suggested that a significant genotypic variation in fatty acid composition of fenugreek genotypes may utilize in developing new cultivars and to introduce fenugreek seed oil for end-uses either sole use or blended with other vegetable oil.