Zoliflodacin is a spiropyrimidinetrione antibiotic that acts by binding to the GyrB part of the DNA gyrase enzyme in bacteria. Its effectiveness for the treatment of Neisseria gonorrhoeae infections has been investigated extensively. Since antibiotic resistance has been reached an alarming rate worldwide, researches on new antimicrobials are considered a priority, especially in the treatment of multidrug-resistant Gram-negative bacteria, such as Klebsiella pneumonia. The aim of this study is to test and compare the effectiveness of zoliflodacin with some traditional antibiotics which are frequently preferred in the treatment of Gram-negative pathogens, primarily K. pneumonia. Additionally, its ability to prevent biofilm formation has also been determined. The minimum inhibitory concentration (MIC) values of zoliflodacin along with levofloxacin, meropenem, gentamicin, ampicillin/sulbactam and ceftazidime/avibactam were evaluated by broth microdilution method against 15 Gram-negative clinical isolates and three standard strains. Also, the synergism potential of zoliflodacin with other antibiotics was evaluated by the checkerboard method against standard strains of K. pneumonia, Pseudomonas aeruginosa, and Acinetobacter baumannii. In addition, the inhibitory effects of zoliflodacin on biofilm formation of standard strains were determined. Zoliflodacin MICs were found to be in the range of 2-64µg/mL, and its combination with meropenem and ampicillin/sulbactam was found to be synergistic, especially against A. baumannii. Zoliflodacin significantly inhibited A. baumannii biofilm at sub-MIC values. These results indicated that zoliflodacin can be considered as an alternative against infections of Gram-negative pathogens, alone or in combination.