Fresh, senesced, and decaying pine needles from Pinus halepensis were evaluated for their allelopathic potential on Festuca arundinacea, Cynodon dactylon and the biosensor plants Avena sativa and Lemna minor through in vivo and in vitro studies. The in vivo study was performed in growth chambers, using 6, 12, and 18 g of pine needle tissue mixed with screened perlite as a substrate. The effects of the different pine needle types were evaluated by determining the total root length, total root surface, root dry weight, total shoot length, total shoot surface, and shoot dry weight. The in vitro study was performed in Petri dishes where seeds from each species were subjected to an increasing concentration of pine needle extract. The extracts were obtained from pine needle ground tissue that was diluted with water and either shacked at room temperature or placed in water bath at 40 °C for 24 h. The evaluation of the allelopathic potential was performed with the determination of radicle length. The allelopathic potential of the pine needle tissues was confirmed with bioassays using oat (A. sativa) and duckweed (L. minor). The results strongly suggested the allelopathic potential of the pine needle tissue, being more pronounced in the fresh, moderate in the senesced, and low in the decaying pine needles. The allelopathic substances were species-specific, and the inhibition resistance of the species tested followed the order F. arundinacea > C. dactylon > A. sativa. The inhibition of the L. minor suggested that the water soluble phytotoxic compounds were inhibitors of Photosystem II.