Although fluorine is abundant in the earth’s crust, it is scarcely found in biomolecules. Adaptive laboratory evolution (ALE) experiments were conducted to introduce organofluorine into living microorganisms. By cultivating Escherichia coli with fluorinated indole analogs, microbial cells evolved that relinquished their dependence on indole and are instead capable of utilizing either 6-fluoroindole (6Fi) or 7-fluoroindole (7Fi) for growth (TrpS-catalyzed in-situ conversion of fluoroindole into fluorotryptophan (FTrp) and TrpRS-catalyzed incorporation of FTrp in context of protein biosynthesis). Consistent and complete adaptation of microbial populations was achieved and the quantitative proteome-wide replacement of Trp by either 6FTrp or 7FTrp was confirmed by nano-LC-MSMS. The dataset comprises the ancestral strain TUB00, two positive controls W-TUB165 and Ind-TUB165 (adapted to grow on tryptophan and indole), three 6Fi adapted lineages 6TUB128-OC, 6TUB165-MB4, 6TUB165-MB3 and two 7Fi adapted lineages 7TUB165-OC and 7TUB165-MB.