Backgrounds: The development of cancer resistance to standard therapy and toxic side effects are the major challenges encountered in chemotherapy treatment of various cancer types. Drugs directly targeting glycolytic enzymes, such as clotrimazole (phosphofructokinase inhibitor), are specific for cancer cells with minor effects on normal cells, due to metabolic difference between normal and cancer cells which depends on aerobic glycolysis to generate ATP. Purpose: The aim of our study was to determine the antiproliferative, anti-migration and anti-invasion effects of clotrimazole on A549 human lung carcinoma and Caco-2 human colorectal adenocarcinoma cells. Methods: To evaluate anticancer potential of the clotrimazole, the methods measuring various antiproliferative endpoints were employed including MTT assay, dynamic monitoring of cell proliferation, invasion and migration with xCELLigence DP Real Time Cell Analyzer. The cytotoxic effects of different concentrations had been investigated for 24 hours on both cells and IC50 values were calculated. Cell proliferation assay were performed with IC50 concentrations (27,5 ?g/mL for Caco-2 and 24,5 ?g/mL for A549 cells). Subsequently, cell migration and invasion analysis were conducted on A549 and Caco-2 cells with the IC50 concentrations. Results: Clotrimazole was significantly induced cell death of both A549 and Caco-2 cells and also IC50 concentrations of clotrimazole were significantly inhibited antimetastatic activity of A549 and Caco-2 cells. Conclusions: In conclusion, this study provides preliminary evidences that clotrimazole might also have significant potential for lung and colon cancer chemotherapy applications.