Colchicine (COL) shows strong anticancer activity but due to its toxicity towards normal cells its wider application is limited. To address this issue, a library of 17 novel COL derivatives, namely N-carbamates of N-deacetyl-4-(bromo/chloro/iodo)thiocolchicine, has been tested against two types of primary cancer cells. These included acute lymphoblastic leukemia (ALL) and human breast cancer (BC) derived from two different tumor subtypes, ER+ invasive ductal carcinoma grade III (IDCG3) and metastatic carcinoma (MC). Four novel COL derivatives showed higher anti-proliferative activity than COL (IC50 = 8.6 nM) towards primary ALL cells in cell viability assays (IC50 range of 1.1-6.4 nM), and several were more potent towards primary IDCG3 (IC50 range of 0.1 to 10.3 nM) or MC (IC50 range of 2.3-9.1 nM) compared to COL (IC50 of 11.1 and 11.7 nM, respectively). In addition, several derivatives were selectively active toward primary breast cancer cells compared to normal breast epithelial cells. The most promising derivatives were subsequently tested against the NCI panel of 60 human cancer cell lines and seven derivatives were more potent than COL against leukemia, non-small-cell lung, colon, CNS and prostate cancers. Finally, COL and two of the most active derivatives were shown to be effective in killing BC cells when tested ex vivo using fresh human breast tumor explants. The present findings indicate that the select COL derivatives constitute promising lead compounds targeting specific types of cancer.