Conjugating a siderophore to an antibiotic is a promising strategy to overcome the permeability-mediated resistance of Gram-negative pathogens. On the basis of the structure of BAL30072, novel pyridone-conjugated monosulfactams incorporating diverse substituents into the methylene linker between the 1,3-dihydroxypyridin-4(1H)-one and the aminothiazole oxime were designed and synthesized. Structure-activity relationship studies revealed that a variety of substituents were tolerated, with isopropyl (compound 12c) and methylthiomethyl (compound 16a) showing the best efficacy against multidrug-resistant (MDR) Gram-negative pathogens. In addition, compound 12c exhibits a good free fraction rate in an in vitro human plasma protein binding test, along with a low clearance and favorable plasma exposure in vivo. In a murine systemic infection model with MDR Klebsiella pneumoniae, compound 12c shows an ED50 of 10.20 mg/kg. Taken together, the results indicate that compound 12c is a promising drug candidate for the treatment of serious infections caused by MDR Gram-negative pathogens.