Bacterial diseases are among the most important causes of losses among fish stocks in the aquaculture industry, affecting the economic development of the sector in many countries. The ability of bacteria to cause disease depends to a large extent on the expression of virulence factors, which help them to invade the host, produce pathological effects and evade host defences. The study of these factors is essential for the development of new immunoprophylactic and chemotherapeutic reagents to fight the bacterial infections, since the development of antibiotic resistance by bacteria has led to these diseases becoming one of the major problems in the sector. In the last decade, the application of in vivo and in vitro molecular techniques to fish pathogenic bacteria, together with the availability of adequate models for studying the disease, have allowed the discovery and characterization of novel virulence determinants, as well as a deeper insight into well-known pathogenic mechanisms. In reference to bacterial diseases, Gram-negative bacteria have long been recognized as one of the main problems in the aquaculture industry. They can cause systemic infections in which they invade the fish and damage internal organs or can cause external infections affecting the gills or causing fin rot and body ulcers. This review describes current understanding of the virulence factors shown to be involved in the virulence of Gram-negative bacteria causing disease in fish.