Chromium (Cr) is one of the most prevalent heavy metals that pose a significant threat to the ecosystem. Its detrimental effects on numerous plant physiological and metabolic pathways subsequently impact growth and development. Employing a combination of glycine betaine (GB) and arbuscular mycorrhizal fungi (AMF) in fenugreek to mitigate Cr toxicity has not been previously investigated in terms of genetics and ultrastructural parameters. Consequently, GB (50 mM) and AMF were selected as ameliorating agents of Cr stress-inducing growth, physiological, ultrastructural, and genotoxicity in fenugreek plants. Through our results, it is obvious that Cr dramatically affects all growth and physiological parameters. At the ultrastructural level, transmission electron microscope (TEM) micrographs indicated a decline in chloroplasts number, disorganization of thylakoids, and fragmentation of chloroplasts, in addition to the accumulation of electrodense materials in the cell wall and inside chloroplasts in Cr-stressed fenugreek leaf cells. However, these abnormalities were relatively restored with AMF and GB applications. The comet assay validated the DNA damage caused by Cr, as indicated by the increased proportion of tailed cells (19.26% ± 1.1), which had mean tail lengths of 12.55 ± 0.96 μm, average DNA content in the tail of 9.28 ± 0.93, and the longest tail moment of 1.07 ± 0.10. In comparison, the control root cells exhibited 6.76 ± 0.25 tailed cells. In contrast to the Cr-stressed group, the combined application of GB and AMF decreased the percentage of tailed cells by 38.93%. Collectively, it was concluded that GB and AMF have a synergistic effect, providing the plants with higher stress resilience.