Limited phytodiversity and regeneration rates occur in some of the southern Brazilian formations known as the Myrtacean Woodlands. Data on phytotoxicity, chemical composition, and allelopathic potential of Myrciaria cuspidata O. Berg, a dominant species in such woodlands, is missing. In this study, both the chemical composition and phytotoxic activity of an aqueous extract (AE) from M. cuspidata leaves were investigated. Target plants were the model species Lactuca sativa L. and the weed Bidens pilosa L. Germination rates, seedling growth, and phenotypic responses of target species were assessed following AE application to determine the inhibitory capacity of M. cuspidata leaf extract. Germination of L. sativa was reduced and delayed in the presence of AE. Strong inhibition of germination was recorded in B. pilosa achenes under the same treatment. Pre-germinated seedlings of L. sativa were essentially not affected by AE, whereas those of the weed showed some negative developmental responses. Overall, inhibitory responses were consistent both in vitro and in soil substrate. Detrimental effects were most apparent in roots and included tip darkening and growth anomalies often preceded by loss of mitochondrial viability. AE proved rich in phytotoxic phenolic compounds including quercetin, gallic and tannic acid. To sum up, AE shows potential as an environmentally friendly pre-emergence bioherbicide of low residual effect and minor environmental impact. Experimental data in laboratory conditions were consistent with potential allelopathic activity of this tree, as inferred from field observations of dominance in the Myrtaceae Woodlands.