This study evaluated the effects of Trigonella foenum-graecum L. (fenugreek) seed extract on stallion spermatozoa quality in vitro, focusing on motility, vitality, and functional competence. In vitro spermatozoa analyses showed that fenugreek seed extract did not demonstrate overt cytotoxicity under the tested conditions: S1 = 1562 µg/mL; S2 = 781 µg/mL; S3 = 390.5 µg/mL; S4 = 195.25 µg/mL; S5 = 97.62 µg/mL; S6 = 48.81 µg/mL; S7 = 24.40 µg/mL. Computer-assisted semen analysis (CASA) revealed a concentration- and time-dependent Treatment × Time interaction for several kinetic parameters. Total motility declined similarly across all groups over incubation time and was not significantly affected by extract concentration, whereas progressive motility, curvilinear velocity, beat-cross frequency, and amplitude of lateral head displacement were selectively increased at specific concentrations (mainly S4 and S7) at the last time point. Metabolic activity (MTT assay) increased significantly relative to the positive control across most concentrations, especially at early incubation time points, with fewer concentrations remaining elevated later. Sperm non-viability (eosin–nigrosin staining) and proAKAP4 levels were also significantly influenced by treatment in a non-monotonic manner: the highest concentration (S1) showed lower non-viability; similarly, the highest concentrations (S1–S3) showed reduced proAKAP4, whereas intermediate-to-lower concentrations showed higher non-viability (mainly S3, S4, S6, and S7) and increased proAKAP4 (mainly S4, S6, and S7), relative to the positive control. Physicochemical parameters of the extract (pH, osmolality) remained within physiological ranges, excluding osmotic or pH-related effects. Fenugreek seeds showed measurable antioxidant activity, with the ABTS assay showing the highest radical-scavenging capacity (10.67 ± 0.28 µmol Trolox equivalents/g dry weight). These findings indicate that fenugreek extract exerts selective, concentration- and time-dependent modulatory effects on stallion sperm function in vitro.