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Shoots of alfalfa (Medicago sativa L.) contain water‐soluble chemical compounds which are autotoxic, i.e., inhibit the growth of alfalfa itself. The objectives of this study were to (i) determine the inhibition threshold of the water‐soluble chemical compounds (autotoxic principle), and (ii) demonstrate that the inhibition of seed germination on early seedling growth of alfalfa is due to autotoxinic compounds from alfalfa shoots and not from microbes. A farmer must make a decision on whether or not it would be advisable to replant alfalfa based on the level of alfalfa residue still growing in the field. Laboratory and greenhouse studies were conducted to determine if autotoxicity in alfalfa due to water‐soluble compounds is concentration dependent. Filter‐sterilized and non‐filter‐sterilized shoot aqueous extracts from vegetative and reproductive stages of ‘WL‐316’ alfalfa were assayed at 20, 40, 60, and 80 g L−1 (fresh shoot weight basis) for their effect on seed germination and root and shoot elongation of seedlings of WL‐316 alfalfa in a growth chamber. Compared with the control, root length, shoot length, and germination were inhibited beyond 20 g L−1 concentration. Shoot extract from the reproductive stage was more inhibitory than from the vegetative stage under laboratory conditions. In the greenhouse, incorporation of 4‐wk‐old green herbage from vegetative stage beyond 48 shoots per square meter level resulted in severe reductions in seedling emergence and plant fresh weight per unit area. Among the several phenolic compounds assayed for their phytotoxicity on root and shoot growth of alfalfa, coumarin and transcinnamic acid at 60 ± 10 μg mL−1 were the most inhibitory. Mixtures of five or more phenolic acids were more phytotoxic than their respective individual components except in the case of trans‐cinnamic acid and coumarin. Autotoxicity in alfalfa may be caused by an interaction of many, yet uncharacterized chemical compounds present in shoots.