Trigonelline has been known as an anti-diabetic substance extracted from plant sources. The Trigonelline content and growth factors of two Trigonella foenum ecotypes (Hamedan and Bandarabbas) treated by two Trichoderma harzianum isolates (chit4215MK and T8-7MK) were studied in a greenhouse. Different growth factors were observed in two ecotypes affected by Trichoderma strains. Hamedan ecotype had more stem length and the Bandarabbas ecotype showed more lateral branches and pod numbers. The results indicated that the Trichoderma strains had a positive effect on the growth of the Hamadan ecotypes stem, and the T8-7MK strain showed better effects than the chit4215MK. No shoot length differences were observed in the Bandarabbas ecotype between the control and Trichoderma treated plants. No significant difference was observed in peroxidase activity and total soluble carbohydrate content between the ecotypes, treatments, and the interaction effects of treatment and ecotypes. The highest Trigonelline content (4 mg g-1 DW) was obtained in the Bandarabbas ecotype treated with the Trichoderma strain T8-7MK, which was 1.6-fold higher than the control plants. Also, the Hamedan ecotype treated by the Trichoderma strain chit4215MK produced a higher content of Trigonelline (3.5 mg g^-1 DW) which was 1.3-fold more than the control plants. The amount of Trigonelline in the treated Hamedan ecotype was lower than the treated Bandarabbas ecotype. Our results revealed that Fenugreek growth factors and Trigonelline biosynthesis can be affected by Trichoderma strains.