Understanding intracellular antibiotic targeting and the associated mechanisms leading to bacterial growth inhibition has been a difficult problem. Here, we discovered the additional intracellular targets of the novel “evolution-drug” lead CD15-3 designed to delay the emergence of antibiotic resistance by inhibiting bacterial DHFR and its Trimethoprim resistant variants. Overexpression of DHFR only partially rescued inhibition of E. coli growth by CD15.3 suggesting that CD15.3 also inhibits a non-DHFR target in the cell. We utilized untargeted global metabolomics and the metabolic network analysis along with structural similarity search of the putative targets to identify the additional target of CD15-3. We validated in vivo and in vitro that besides DHFR CD15-3 inhibits HPPK (folK), an essential protein upstream of DHFR in bacterial folate metabolism. This bivalent cellular targeting makes CD15-3 a promising lead to develop a “monotherapy analogue” of combination drugs.Competing Interest StatementThe authors have declared no competing interest.