We attempted to generate de novo resistance to a newly described biocidal complex, ITC (iodo-thiocyanate complex), and to levofloxacin (LVX) in Escherichia coli ATCC 25922, by means of selective chemostat culture. We measured resistance by determining the minimum inhibitory concentrations (MICs) for these agents. E. coli underwent 20-day parallel adaptive evolution routes under no antimicrobial selection, and gradually increasing ITC and LVX selection pressure. Long-term exposure of E. coli to ITC did not induce resistance to ITC, or cross-resistance to LVX. No distinct mutational pattern was evidenced from whole-genome sequence (WGS)-based comparisons of ITC-challenged and unchallenged bacterial populations. Moreover, the exposed E. coli population could not survive a 2 × MIC challenge of ITC. By contrast, resistance to LVX was rapidly induced (on day 1 the MIC had increased 16-fold), selected for (by day 14 the MIC had increased 64-fold) and enriched with a highly characteristic genome mutational pattern. WGS of this evolving population revealed that the majority of mutations appeared in the genes of LVX target proteins (GyrA, ParC, ParE) and drug influx (OmpF). This study suggests that the usage of ITC may not trigger the emergence of facile resistance or cross-resistance, in contrast to common antibiotics.