There is an urgent need for new antimicrobials to treat the opportunistic Gram-negative , which represents a problematic challenge for cystic fibrosis patients. Recently, a benzothiadiazole derivative, C109, was shown to be effective against the infections caused by and other Gram-negative and-positive bacteria. C109 has a promising cellular target, the cell division protein FtsZ, and a recently developed PEGylated formulation make it an attractive molecule to counteract infections. However, the ability of efflux pumps to extrude it out of the cell represents a limitation for its use. Here, more than 50 derivatives of C109 were synthesized and tested against Gram-negative species and the Gram-positive . In addition, their activity was evaluated on the purified FtsZ protein. The chemical, metabolic and cellular stability of C109 has been assayed using different biological systems, including quantitative single-cell imaging. However, no further improvement on C109 was achieved, and the role of efflux in resistance was further confirmed. Also, a novel nitroreductase that can inactivate the compound was characterized, but it does not appear to play a role in natural resistance. All these data allowed a deep characterization of the compound, which will contribute to a further improvement of its properties.Copyright © 2020 Chiarelli, Scoffone, Trespidi, Barbieri, Riabova, Monakhova, Porta, Manina, Riccardi, Makarov and Buroni.