The effects of storage time on Eriochloa villosa (Thunb.) Kunth seed longevity and germination were investigated. A number of physiological and biochemical indexes, such as germination indexes, seed viability, storage materials, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and α-amylase (AMS) activity, were determined to investigate the mechanisms behind E. villosa seed longevity. The longevity of E. villosa seeds under indoor dry storage conditions was more than eight years. The vitality of E. villosa seeds decreased dramatically with increasing storage time. The malondialdehyde (MDA) concentration of the seeds increased dramatically with an increase in the storage period. The SOD, POD, CAT, and AMS activities significantly decreased over time, while the soluble sugar content first increased and then decreased. Storage duration significantly affected the soluble protein content of E. villosa seeds. The germination index of the seeds correlated with their physiological and biochemical indices and showed a significant positive correlation with the soluble sugar content, SOD, POD, and AMS activity, and a significant negative correlation with the MDA concentration. This study is of great importance for understanding the characteristics of the field seed bank of E. villosa and for implementing integrated weed control measures to contribute to sustainable agricultural development.