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Serotonin (SER) and melatonin (MEL) are signalling molecules involved in signalling in various stress response mechanisms in plants. However, their role and molecular mechanism in the accumulation of isoflavones and biomass under temperature stress are not clearly defined. To explicate their function, SER and MEL at different ratios were applied to the in vitro cultures of soybean at 16, 24, and 32 °C and were analyzed for the accumulation of bioactive compounds, isoflavones content, and culture biomass followed by transcript levels of cell division, isoflavones, and ethylene biosynthesis genes. An increase in biomass, total phenolics, total flavonoids, and different forms of isoflavones content was evident in the treatments. The expression level of critical genes of isoflavone (chalcone synthase, chalcone reductase, chalcone isomerase, and isoflavone synthase), ethylene (s-adenosyl methionine and 1-aminocyclopropane-1-carboxylic acid oxidase), cell division (cyclin and cyclin-dependent kinase), and MEL biosynthesis genes (acetyl serotonin O-methyltransferase and serotonin N-acetyltransferase) and transcription factors (melatonin 2 hydroxylase, myeloblastosis like protein) were co-expressed during temperature stress in the presence of SER and MEL. At 32 °C, the SER and MEL application showed maximum biomass and isoflavones content and was substantiated with the corresponding gene expression. We hypothesize that the SER and MEL ratio may play a role in increasing the biomass and isoflavones content under temperature stress in soybean cell culture.