Flavobacterium spp. infections remain a major aquaculture industry issue and can cause major losses to fish stocks and economic implications. For salmonid producers, a common threat within this genus is F. psychrophilum, the causative agent of bacterial coldwater disease (BCWD). Emerging family Flavobacteriaceae isolates have also been identified in global fish health diagnostic cases and include novel, virulent Flavobacterium and Chryseobacterium species. A live-attenuated immersion vaccine (B.17-ILM), developed at the University of Idaho, has been shown to confer significant protection to salmonids. To further characterize cross-protective efficacy of this live attenuated vaccine, a series of vaccination/challenge trials were carried out. In the first trial (Trial 1), this immersion vaccine was tested (in rainbow trout) against 9 virulent F. psychrophilum isolates in comparison with a domestic, wild-type virulent strain. In a follow-up trial (Trial 2), rainbow trout were then challenged with two Chryseobacterium spp. (S25 and T28), a Flavobacterium spp. (S21), a mixed combination of S21:S25:T28 strains, and a standard virulent F. psychrophilum CSF259-93 strain. Across both trials, vaccinated fish developed an adaptive immune response (as measured by F. psychrophilum-specific antibodies) that increased out to the time of challenge (8 weeks post-immunization). Results of Trial 1 showed that the vaccine provides significant protection against all F. psychrophilum strains tested, with relative percent survival (RPS) values ranging from 51-72%. In Trial 2, the CSF259-93 challenged group had a RPS of 94.44% compared to the unchallenged control group. Interestingly, vaccinated fish also exhibited significantly lower cumulative mortality and a high RPS (85.18%) following the mixed family Flavobacteriaceae infection, but protection was not observed following challenge with the single family Flavobacteriaceae strains. Results suggest that this live attenuated vaccine will elicit a protective immune response to fish and provide a valuable tool for BCWD control even in aquaculture operations affected by diverse strains of F. psychrophilum and/or mixed infections with novel family Flavobacteriaceae strains.