Drug efficacy determined in preclinical research is difficult to transfer to clinical practice. This is mainly due to the use of oversimplified models omitting the effect of the tumor microenvironment and the presence of various cell types participating in the formation of tumors in vivo. In this study, we used robust three-dimensional models including spheroids grown from colon cancer cell lines and organotypic cultures prepared from the colorectal carcinoma tissue to test novel therapeutic strategies. We developed a multi-modal approach combining brightfield and fluorescence microscopy for evaluating drug effects on organotypic cultures. Combined treatment with 5-fluorouracil and disulfiram/copper efficiently eliminated cancer cells in these 3D models. Moreover, disulfiram/copper down-regulated the expression of markers associated with 5-fluorouracil resistance, such as thymidylate synthase and CD133/CD44. Thus, we propose combined therapy of 5-fluorouracil and disulfiram/copper for further testing as a treatment for colorectal carcinoma. In addition, we show that organotypic cultures are suitable models for anti-cancer drug testing.