Depleting the soil weed seedbank is an important integrated weed management strategy that has the potential to foster lasting weed control. Long-term dormancy and decay resistance of weed seeds pose a challenge to weed eradication efforts. Select soil fungi have been shown to cause significant decay of weed seeds. The physical and chemical mechanisms by which seeds in the seedbank defend themselves against pathogens have been well researched. However, very few studies have purposefully investigated the biochemical defence response of seeds. Enzyme-based biochemical seed defences have been detected in dormant and non-dormant seeds, and research supports their function in pathogen defence. This review summarizes current knowledge of the seed defence enzymes polyphenol oxidase, peroxidase, chitinase and oxalate oxidase. The fungal enzymes chitinase, protease and xylanase that function in pathogenesis of seeds in the soil seedbank are also reviewed. Progress in the development and standardization of in situ enzyme analyses fosters our understanding of actual enzyme activity present in soils, while high-throughput microplate techniques promote efficiency and enable greater scope. Application of genomic, proteomic and transcriptomic techniques to glean a deeper and more holistic understanding of the enzymatic interactions of weed seeds and soil fungi in the soil seedbank will support the development of improved integrated weed management strategies.