The continued emergence of multi-antibiotic-resistant strains of Neisseria gonorrhoeae necessitates the development of new antibiotics that are effective against this human pathogen. We previously described that the RNA polymerase-targeting antibiotic corallopyronin A (CorA) has potent activity against a large collection of clinical strains that express different antibiotic resistance phenotypes including when such gonococci are in a biofilm state. Herein, we tested whether a CorA-sensitive gonococcal strain could develop spontaneous resistance. Our finding that CorA resistance could only be achieved by a multi-step process involving over-expression of the MtrCDE efflux pump and single amino acid changes in RpoB and RpoC suggests that such resistance may be difficult for gonococci to evolve if this antibiotic is used in the future to treat gonorrheal infections that are refractory to cure by other antibiotics.