The aim of this study was to determine the effect of the hydrolates obtained as the by-products of the Thymus vulgaris essential oil steam distillation process. The bioassays, which were undertaken in order to determine the effect on germination and initial growth of seedlings of some cultivated and weed species, were performed under controlled conditions with different concentrations of the hydrolates. Seeds of Glycine max, Helianthus annuus, Zea mays, Triticum aestivum, Daucus carota subsp. sativus, Allium cepa, Medicago sativa, and Trifolium repens, and six weed species—Amaranthus retroflexus, Chenopodium album, Portulaca oleracea, Echinochloa crus-galli, Sorghum halepense, and Solanum nigrum—were treated with 10, 20, 50, and 100% T. vulgaris hydrolate solution. The obtained results showed that the T. vulgaris hydrolate had the least negative effect on the germination of cultivated species, such as soybean, sunflower and maize, whereas clover and alfalfa were the most sensitive. By comparison, all the tested weed species expressed high susceptibility. It can be concluded that the T. vulgaris hydrolate has an herbicidal effect, in addition to its potential as a biopesticide in terms of integrated weed management.