Maize roots can host free-living nitrogen-fixing bacteria (diazotrophs), but their agronomic potential remains underutilized. A study with two maize inbred lines (FV2, FV252) inoculated with Herbaspirillum seropedicae (SmR1, SmR54) and Azospirillum brasilense (FP2, FP10) showed differential colonization. FV2 displayed significantly higher N?-fixation than FV252, confirmed by acetylene reduction assay (effective within 7–14 days). Inoculation with nitrogenase-active strains altered root and leaf metabolites, as revealed by metabolomic profiling. Endophytic H. seropedicae cells were quantified by a specific protocol, while confocal microscopy (SmR1-RAM10 strain) confirmed colonization. Data were clustered and analyzed using t-tests in MeV software (v4.9). Findings highlight strain- and genotype-dependent N?-fixation efficiency and metabolic changes, offering insights into optimizing maize–diazotroph associations for sustainable nitrogen input.