Iron is an essential micronutrient for nearly all microorganisms, yet its bioavailability is severely limited in most environments. To overcome this restriction, microorganisms produce siderophores, high-affinity iron-chelating molecules that play pivotal roles in microbial iron homeostasis, interspecies competition, and host–pathogen interactions. Despite extensive research, current understanding of siderophore biosynthetic and regulatory diversity remains largely limited to specific models, with comprehensive cross-taxonomic frameworks only beginning to emerge. This review systematically integrates recent advances in the genetic and biochemical foundations of microbial siderophore production, focusing on the two major biosynthetic pathways: nonribosomal peptide synthetase (NRPS)-dependent and NRPS-independent synthetase (NIS). We further elaborate on the diverse transport systems in Gram-negative and Gram-positive bacteria, as well as fungi, alongside the iron-responsive regulators (e.g., Fur) and gene clusters that coordinate iron uptake and utilization. Beyond physiological mechanisms, we discuss how these insights inform emerging applications of siderophores across multiple fields: in medicine, enabling “Trojan horse” antimicrobial strategies; in agriculture, enhancing plant iron uptake and serving as biocontrol agents; in environmental remediation, facilitating heavy-metal detoxification; and in biosensing, acting as selective probes for metals and pathogens. By bridging fundamental mechanisms with practical applications, this review aims to provide an integrative perspective for future exploration of microbial iron homeostasis and its biotechnological potential.