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Secondary metabolites (SMs) have high economic impact thanks to their exploitability in chemical, pharmaceutical and cosmetic industries. Trigonella foenum-graecum has an importance due to the production of bioactive compounds with pharmaceutical values. Among them, the alkaloid trigonelline is known for its role in the treatment of different human diseases. SM accumulation is influenced by environmental factors but is modulated by the application of exogenous compounds. Ethephon, a precursor of the phytohormone ethylene, was already used to influence SM accumulation. Our work is aimed at evaluating the accumulation of trigonelline and the phytohormone abscisic acid (ABA) when three factors were combined: i) two levels of water regimes (well-watered and water deficit), ii) ethephon treatments and iii) inoculation with an arbuscular mycorrhizal (AM)-based inoculum also leading to nodulation. The content of trigonelline and ABA was significantly affected by symbioses, showing high accumulation in AM-colonized plants irrespective of the water regimes applied. In terms of trigonelline accumulation with respect to ethephon treatments, while symbiotic plants showed a dose-dependent trend, non-symbiotic plants showed a significantly difference only when 550 ppm of ethephon was applied. In conclusion, our work provides new information on the effects of both ethephon and symbioses on plant growth and accumulation of valuable compounds, such as trigonelline, in fenugreek.