Among abiotic stresses in plants, drought and chilling stresses reduce the supply of moisture to plant tissues, inhibit photosynthesis, and severely reduce plant growth and yield. Thus, the application of water stress-tolerant agents can be a useful strategy to maintain plant growth under abiotic stresses. This study assessed the effect of exogenous bio-based 2, 3-butanediol (BDO) application on drought and chilling response in tomato and turfgrass, and expression levels of several plant signaling pathway-related gene transcripts. Bio-based 2, 3-BDOs were formulated to levo-2, 3-BDO 0.9% soluble concentrate (levo 0.9% SL) and meso-2, 3-BDO 9% SL (meso 9% SL). Under drought and chilling stress conditions, the application of levo 0.9% SL in creeping bentgrass and meso 9% SL in tomato plants significantly reduced the deleterious effects of abiotic stresses. Interestingly, pretreatment with levo-2, 3-BDO in creeping bentgrass and meso-2, 3-BDO in tomato plants enhanced JA and SA signaling pathway-related gene transcript expression levels in different ways. In addition, all tomato plants treated with acibenzolar-S-methyl (as a positive control) withered completely under chilling stress, whereas 2, 3-BDO-treated tomato plants exhibited excellent cold tolerance. According to our findings, bio-based 2, 3-BDO isomers as sustainable water stress-tolerant agents, levo-and meso-2, 3-BDOs, could enhance tolerance to drought and/or chilling stresses in various plants through somewhat different molecular activities without any side effects.