There are renewed global calls for the development of new antimicrobial drugs with novel mechanisms of action, which also prevent further resistance to existing classes against multi-drug resistant pathogens. In this study, the potential of a novel chemical analogue of the anticoccidial robenidine (NCL195) as a drug against multi-drug resistant Gram-positive and Gram-negative bacterial infections was investigated. We showed the minimum inhibitory concentration (MIC) range of NCL195 against methicillin-resistant Staphylococcus aureus isolates was 1-2 μg/mL1. NCL195 demonstrated synergistic activity in combination with subinhibitory concentration of colistin against clinical multidrug-resistant Gram-negative bacterial pathogens, with MICs for NCL195 ranging from 0.5-4 μg/mL for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa in the presence of subinhibitory concentrations of colistin, whereas NCL195 alone had no activity2. Transmission electron microscopy of S. aureus cells after treatment with NCL1951, or of E. coli and P. aeruginosa after treatment with NCL195+colistin combination confirmed marked ultrastructural changes in membrane morphology, most frequently in the cell envelope2,3. In bioluminescent mouse sepsis challenge models, administration of four oral doses of 50 mg/kg NCL195 (4 hours apart) resulted in significantly reduced S. aureus loads and longer survival times than vehicle-only treated mice. Furthermore, administration of four oral doses of 50 mg/kg NCL195 (4 hours apart) combined with four intraperitoneal doses (4 hours apart) of colistin (0.125 mg/kg) resulted in significant reduction in E. coli loads compared to treatment with NCL195 alone. In addition, treatment with the NCL195+colistin combination resulted in better E. coli clearance than treatment with colistin alone at the same concentrations. We conclude that NCL195 is a potential alternative for further development for specific treatment of Gram-positive bacterial infection or treatment of Gram-negative infection in combination with subinhibitory concentrations of colistin.