The emergence of antimicrobial-resistant Neisseria gonorrhoeae necessitates improved understanding of physiological factors that influence antimicrobial responsiveness beyond genetic resistance mechanisms. Iron acquisition is essential for gonococcal metabolism, virulence, and biofilm development. However, the impact of iron availability on antimicrobial susceptibility and biofilm stability remains incompletely defined. To evaluate the role of iron availability, antimicrobial susceptibility and biofilm dynamics were assessed under iron-replete and iron-restricted conditions using the iron chelator deferoxamine mesylate (DFO). Minimum inhibitory concentrations (MICs) were determined using agar dilution, broth microdilution, and gradient diffusion (E-test) assays across multiple gonococcal strains, including FA1090, FA19, and an efflux-deficient mutant (RD1). Biofilm formation and stability were evaluated using static microtiter plate assays, crystal violet staining, and colony-forming unit (CFU) enumeration. Iron restriction altered antimicrobial susceptibility across multiple antibiotics and assay platforms, frequently resulting in decreased MIC values under iron-limited conditions. These effects were strain- and antibiotic-dependent and were observed in both wild-type and efflux-deficient backgrounds, indicating that iron availability influences antimicrobial responsiveness independently of classical efflux-mediated resistance. In biofilm assays, iron chelation resulted in a dose-dependent reduction in biofilm biomass, with FA1090 exhibiting greater sensitivity to iron limitation than FA19. Iron restriction also destabilized established biofilms and enhanced antibiotic-mediated reductions in biofilm biomass, particularly at sub-MIC and near-MIC concentrations. Notably, reductions in biofilm biomass did not consistently correspond to reductions in bacterial viability, highlighting distinct effects on biofilm structure and survival. These findings identify iron availability as a key determinant of gonococcal antimicrobial responsiveness and biofilm stability. These results underscore the importance of environmental conditions in shaping antimicrobial activity and provide a framework for understanding how host-imposed nutrient limitation may influence treatment outcomes in N. gonorrhoeae.