Allelopathy is a unique phenomenon in nature that refers to direct or indirect, positive or negative, impact of one plant (including microorganisms, insects, and herbivores) on another plant through chemical secretions (allelochemicals). Ambrosia artemisiifolia (ragweed) is a well-known invasive plant possessing allelochemicals that have been found to inhibit the growth and development of plants in their immediate environment. This study aimed to determine the allelopathic impact of the aqueous leaf extract isolated from ragweed leaves (concentrated extract, 1:2, 1:4, and 1:8) on seed germination and early seedling growth of barley (Hordeum vulgare L.) and white clover (Trifolium repens L.). The examined aqueous extract of ragweed had a higher inhibitory effect on the germination of white clover seeds compared to barley. The germination of barley seeds was the lowest at the concentrated leaf extract (up to 70%), while the same process in barley seeds was not determined at this concentration. In the other treatments, germination was significantly lower in both examined species compared to the control. Shoot and root growth of both researched species was inhibited in all treatments compared to the control. The obtained results indicate that the aqueous ragweed extract significantly inhibits the growth of roots and shoots of both tested species, which increases the need for more intensive research on this topic.