Polyamines are biogenic polycationic molecules involved in key cellular functions. Extracellular polyamines found in bodily fluids or laboratory media can be imported by bacteria or bind to negatively charged bacterial surface structures, where they can impair binding of antimicrobials. We hypothesized that the presence of polyamines in fluids that bathe urogenital mucosal surfaces could alter the susceptibility of the sexually transmitted strict human pathogen Neisseria gonorrhoeae to mediators of the innate host defense. Herein we report that polyamines can significantly increase gonococcal resistance to two structurally diverse cationic antimicrobial peptides (polymyxin B and LL-37) but not to antibiotics that exert activity in the cytosol or periplasm (e.g., ciprofloxacin, spectinomycin, or penicillin). The capacity of polyamines to increase gonococcal resistance to cationic antimicrobial peptides was dose dependent, correlated with the degree of cationicity, independent of a polyamine transport system involving the polyamine permeases PotH and PotI, and was reversible. In addition, we found that polyamines increase gonococcal resistance to complement-mediated killing by normal human serum. We propose that polyamines in genital mucosal fluids may enhance gonococcal survival during infection by reducing bacterial susceptibility to host-derived antimicrobials that function in innate host defense.