Columnaris disease is a pervasive threat to a wide range of wild and cultured freshwater fish worldwide. There are currently no efficacious preventative methods available and most commercially valuable species cultured in the United States are susceptible, including salmonids, catfish (Ictalurus spp.), and tilapia (Oreochromis spp.). Flavobacterium columnare, the historical etiologic agent, has been recently reclassified into four related species: F. columnare, F. covae, F. davisii and F. oreochromis. In salmonids, F. columnare and F. davisii are the main agents associated with outbreaks in both wild and captive populations. We hypothesized that accumulation of mutations in F. columnare and F. davisii by serial passage in the presence of rifampin can generate immunogenic live attenuated vaccines (LAVs) that confer protection to vaccinated fish challenged with heterologous Columnaris causing-agents. Four lineages of rifampin-resistant strains were generated from two genetically distinct parent strains (n=4) by multiple passages in increasing increments of Rifamycin SV sodium salt. Attenuation was initially evaluated quantifying cytotoxicity in an CHSE cell line comparing the passaged and parental strains. Chinook salmon and rainbow trout challenges were then used to assess strain virulence and the protection conferred by LAV candidates against virulent F. columnare. Multiple passaged strains demonstrated significant reductions in cytotoxicity in CHSE (p < 0.05). Selected strains showed attenuated virulence in the salmonid challenge model. Immunization by immersion of some candidates proffered protection against a virulent WT strain of F. columnare; however, standardization of vaccination protocols are still needed. In conclusion, passaging of F. davisii in increasing concentrations of antibiotic is a viable method for generating LAV candidates with the potential to protect against heterologous WT strains of Columnaris causing-agents in salmonids.