Synthesis of novel 4-aminoquinoline-rhodanine hybrid using inexpensive starting materials via easy to operate methodology, and their biological activity is reported. All the compounds were screened for their in vitro antimalarial activity against chloroquine-resistant (K1) and chloroquine-sensitive (3D7) strains of Plasmodium falciparum, and their cytotoxicity toward VERO cell line. Compounds 9, 19, 21 and 23 exhibited excellent antimalarial activity with IC50 value ranging from 13.2 to 45.5 nM against chloroquine-resistant (K1) strain. Biochemical studies revealed that inhibition of hemozoin formation is the primary mechanism of action of these analogs for their antimalarial activity. Additionally, some derivatives (14, 18 and 26) of this series also exhibited the antimycobacterial activity against H37Rv strain of Mycobacterium tuberculosis with MIC value of 6.25 μM.