Antibiotics revolutionised modern medicine, not only for treating bacterial infections, but also for ensuring the safety of surgery, organ transplantation, and cancer chemotherapy. However, rising antimicrobial resistance threatens their efficacy in the clinic. Clostridioides difficile and Escherichia coli are important pathogens that present distinct challenges for antibiotic development. Selective activity against C. difficile is needed to avoid disrupting the beneficial gut microbiome, while poor permeability across the Gram-negative cell envelope limits drug development against pathogens such as E. coli. The gut bacterium C. difficile is the most common of all hospital-acquired infections in the USA and places a heavy burden on the healthcare system. Disruption of the microbiome caused by antibiotic use is a major risk factor for infection, where the lost diversity contributes to a high rate of recurrent infection. Selective antibiotics which spare the microbiome are crucial to combating recurrent infection. In Gram-negative bacteria, the Lol (localization of lipoprotein) system is essential for outer membrane biogenesis. The ATP-driven transporter complex LolCDE has emerged as a promising, yet underexplored, antibacterial target. Some LolCDE inhibitors have been reported, but limited structure–activity information is available, and none have yet progressed to clinical trials. In Manuscript I, published microbiome datasets were leveraged to identify fentiazac, a known anti-inflammatory drug, as a selective inhibitor of C. difficile. Structure-activity optimisation identified a compound with increased potency, which selectively inhibited C. difficile in a model gut microbiome and was active against clinical isolates. The compound retained some anti-inflammatory activity and was non-toxic to human cells. Sequencing of resistant mutants proposed DNA polymerase subunit DnaE as the molecular target. In Manuscript II, structure–activity relationships of a pyrazole-based E. coli LolCDE inhibitor were systematically investigated. Structural motifs associated with toxicity and poor metabolic stability were identified, and further optimisation produced analogues with increased potency and improved drug-like properties. In Manuscript III, structural motifs shared by topologically similar LolCDE inhibitors were explored through systematic modification of the core heterocycle. Insights from Manuscript II were then applied to optimise a novel series of triazole-based inhibitors with improved potency and drug-like properties. The three works in this thesis are unified by the overarching societal demand for new starting points to feed into the antibiotic development pipeline. The described compound series provide a solid base for further studies towards antibacterials against C. difficile and priority Gram-negative pathogens.