Gentamicin is an aminoglycoside antibiotic produced by various species of the genus Micromonospora and has received much attention in the recent years as a broad-spectrum antibiotic for treatment of various infections. It exists as a complex of closely related aminoglycoside structures and the clinically significant one is the gentamicin C complex. This review article focuses attention on the present status of knowledge and the main advancements achieved in the last few decades on the subject of gentamicin with regard to its production, biosynthetic pathway, mode of action, and uses. The various nutritional and environmental parameters affecting gentamicin production and the factors affecting the release of bound gentamicin are discussed. Further, strain improvement using UV and/or chemical mutagenesis can be applied to augment the efficiency of the producer strain and a number of case studies are presented. Different detection and quantitative methods for gentamicin estimation and the mode of action of gentamicin are discussed in detail. This antibiotic finds extensive use in combination chemotherapy and as a drug for different delivery agents for treatment of osteomyelitis and other recent applications in gene therapy.