The whitefly, Bemisia tabaci, an insect of the order Hemiptera which attacks more than 600 species of plants, is one of the most important agricultural pests around the world. The insecticidal Cry proteins from Bacillus thuringiensis (Bt) are useful biological pesticides, and some are toxic to Hemipteran insects. In this study, Colombian native isolates of Bt were functionally characterized at molecular and biological levels. The strains contained between one and five different crystal shapes: round, triangular, amorphous, bipyramidal and squared. The strains presented between three to seven bands of proteins in their electrophoretic pattern that were organized into six groups according to their possible biological activity on insect pests. Cry1Aa, cry1Ab, cry1Ac, cry1B and cry1C genes were identified for PCR in the different Bt isolates. Bioassays were performed on tomato leaves whose surface was spread with 3 μg · ml−1 crude extract of Bt toxins. Second instar larvae of whitefly, which were placed on top of leaves and exposed to the toxins for 7 days, exhibited mortalities from 18 to 69%. The lethal concentration 50 (LC50) of ZBUJTL39, Bt kurstaki HD1 and ZCUJTL9 strains were 1.83, 1.85 and 2.16 μg · ml−1, respectively (p < 0.05). These results show that the native Bt strain ZBUJTL39, which contained the genes cry1Aa, cry1Ab, cryCa and cryBa could eventually be used for the development of an integrated management program together with other tools for the control of B. tabaci.