The application of plant growth regulators (PGRs), such as abscisic acid (ABA), putrescine (Put), and 2,4-epibrassinolide (EBR), has been shown to enhance a plant's resistance to various abiotic stresses. However, the protective effects of these PGRs on tomato (Solanum lycopersicum) seedlings under suboptimal temperature stress have not yet been evaluated. We also do not know the most effective method of application of PGRs for various tomato cultivars. We studied the effects of three rates of exogenous ABA, Put, or EBR in limiting damage from suboptimal temperature stress on two tomato cultivars, Zhongshu6 (considered sensitive to suboptimal temperatures) and SANTIAM (considered tolerant to suboptimal temperatures). Results showed that application of these PGRs at appropriate concentrations could effectively reduce the decline in the net photosynthetic rate (Pn) and the chlorophyll (Chl) content in leaves caused by suboptimal temperature stress in both ‘Zhongshu6’ and ‘SANTIAM’ and could promote an increase in organic osmolyte (proline and soluble sugar) contents and root 2,3,5-triphenyltetrazolium chloride (TTC)-reducing activity for ‘Zhongshu6’. However, these effects were inferior on ‘SANTIAM’. For both cultivars, the best treatment concentrations are 1 mm ABA, 0.1 mm Put, or 0.02 μM EBR. Results indicate that in tomato production, exogenous application of ABA, Put, or EBR at appropriate concentrations can effectively limit damage from suboptimal temperature stress.