The latest research has shown that chitosan acts as a growth stimulator and elicitor in plants, including resistance to biotic and abiotic factors. However, increasing concentrations could possibly make chitosan a source of stress for plants. In this study, we investigated the effect of low-molecular-weight chitosan hydrolysate on the root development of tomato (Solanum lycoperscum) cultivars Red Cherry, Lel, and Tytan. The growth rate change, together with total phenolic content, phenylalanine ammonia-lyase (PAL) activity, and gene expression, were studied in relation to tomatoes. High concentrations of chitosan negatively affect the growth of tomato seedlings and contribute to changes in the tropism of the seedling roots. After the addition of chitosan hydrolysate, the PAL activity and the total phenolic content decreased 24 h later. PAL is a key enzyme in the biosynthesis of many plant stress factors. An analysis of the tomato PAL gene family was carried out. The SlPAL gene expression in the seedlings of cv. Cherry increased 1.5 times after 48 h, while in cv. Lel, the expression stably decreased in the presence of chitosan. The obtained results are supposed to aid our understanding of the mechanisms underlying the effects of chitosan on plant development and further its successful application in agriculture as well as in research on plant stress.