Seaweeds, an integral part of marine coastal environment, are an important food, industrial raw materials and biostimulants for crop growth due to the presence of a number of plant growth stimulants. The sap of an economically important seaweed Kappaphycus alvarezii (K-sap) is gaining momentum for sustainable intensification of agricultural productivity. In the present study, the phytohormone levels and transcript regulation of defence-related genes were studied in tomato seedlings in response to K-sap application alone and in combination with the fungus Macrophomina phaseolina, the causal agent of charcoal rot in tomato. The application of Ksap alone and in combination with fungus M. phaseolina significantly increased the concentration of ABA, IAA, SA and zeatin hormones. The enhanced transcript of pathogenesis related genes (PR-1b1, PR-3 and PR-5) was observed in response to application of K-sap alone and in combination with fungus M. phaseolina in tomato. The transcription factor, Pti4 and mitogen-activated kinase pathway gene, MPK2 also showed transcript accumulation on combined treatment of Macrophomina and K-sap. This is the first report highlighting the differential regulation of defence-related genes in accordance with phytohormone levels on application of seaweed sap.