Allelopathy is a well known phenomenon occurring in the rhizosphere and involving plants and other organisms, which consists in a number of stimulatory or inhibitory effects that can play an important role in either natural plant communities or agro-ecosystems. This phenomenon is ascribed principally to secondary products of plant metabolism of wide chemical nature, named “allelochemicals”, which can be identified in plant extracts and root exudates of wild and cultivated tree and herbaceous species. The study of allelochemicals may provide useful information not only for the correct choice of crop rotation, thus avoiding autotoxic problems, but also for their potential use as selective biopesticides. Humic substances, which are the most important components of non-living soil organic matter and are ubiquitous in any natural and cultivated soil, are expected to play a relevant role in plant allelopathy. A recent work demonstrated that soil Humic Acid (HA) and Fulvic Acid (FA) fractions can modulate significantly the allelopathic potential of known phenolic acids for lettuce and tomato seedlings. In this study, various fractions of carrot and tomato root exudates were tested on seed germination and seedling early growth of the same species (autotoxic potential) and lettuce (allelochemical activity). One of these fractions in combination with soil HA or FA was also tested on lettuce. Carrot or tomato plants were grown in hydroponics under controlled climatic conditions in a Phytotron growth chamber for one month. At the end of this period, the nutrient solution was removed and replaced with distilled water. The plants were left in the same pots for two more days, and then the growth medium was collected. Aliquots of 4 L of this solution were evaporated to 200 mL under reduced pressure at 40°C, and then fractionated by using Liquid-Liquid Partitioning (LLP) or Solid Phase Extraction (SPE) techniques. Chloroform (Cl), Buthanol (Bu) and Ethyl Acetate (EA) extracts of root exudates were obtained by LLP, and Acetonitrile (ACN) and Methanol (Me) extracts by SPE. An aliquot of each extract was dried, and the residue dissolved in the same volume of an aqueous acetone solution (0.2%). These solutions (Cl, Bu, EA, ACN and Me fractions) were tested for seed germination and seedling early growth (6 day-growth for tomato and carrot and 4 day-growth for lettuce). The Cl fraction from tomato root exudates at two concentrations was also tested in combination with a soil HA or FA at a concentration of 200 mg/L. In general, all fractions from carrot and tomato root exudates inhibited significantly the germination and primary root elongation of lettuce. The greatest reductions of lettuce root lengths were observed in the treatments with the Bu fraction from carrot (46% of the control) and the Me fraction from tomato (33% of the control). Generally, the root exudate fractions from both species affected lettuce shoots less than roots, and at an extent depending on the type of fraction. The greatest inhibitory effect was measured in the treatments with the Cl fractions from both species. A slight autotoxic activity was observed in the case of tomato, which was produced especially by the Me fraction and mainly on root elongation, whereas no autotoxic activity was observed for carrot. Further, the Cl fraction from tomato root exudates applied at the higher concentration decreased lettuce root and shoot length of about 20 and 46%, respectively, and showed a greater negative effect when applied in combination with HA or, especially, FA. In particular, the presence of HA increased the inhibitory effects of the Cl fraction only on shoot elongation, whereas the presence of FA caused a greater decrease of both shoot and root lengths. These results indicate the significant role of humic substances in the regulation of the allelophatic phenomenon.