A broad spectrum biomolecule Hapalindote-T, from a cyanobacterium Fischerella sp. colonizing Neem tree bark was used for its targets using Escherichia coli. The cellular extracts of Hap-TS (sensitive) and Hap-TR (resistant) of E. coli were subjected to 2DGE. The protein spots (selected) with altered expression were analysed by LC-MS. The data obtained was matched with database of E. coli. Seventeen proteins were found with altered expression level. Three membrane proteins, OmpP, Agn43A and LysU, found in Hap-TS strain were absent in the Hap-TR strain. However, fourteen proteins, AspA, GlpK, LpdA, HslU, GlnA, SucB, YihT, GalF, MDH, RfbB, RmlB, AcrAB, FabB and GapA, related to certain metabolic pathways of the cell and overproduced in the extract of Hap-TR strain. The seventeen screened proteins were related with vital metabolic pathways including membrane protein (Omp P), in E. coli. The results indicated that these proteins might be the cause of resistance in E. coli. These results suggested that overproduced proteins/enzymes in the resistant strain might be a survival strategy under Hap-T stress and could be used as signature protein for the development of new drugs.