Rainbow trout fry syndrome (RTFS) and bacterial coldwater disease (BCWD) is a globally distributed freshwater fish disease caused by Flavobacterium psychrophilum. In spite of its importance, an effective vaccine is not still available. Manipulation of the microbiome of skin, which is a primary infection gate for pathogens, could be a novel countermeasure. For example, increasing the abundance of specific antagonistic bacteria against pathogens in fish skin might be effective to prevent fish disease. Here, we combined cultivation with 16S rRNA gene amplicon sequencing to obtain insight into the skin microbiome of the rainbow trout (Oncorhynchus mykiss) and searched for skin bacteria antagonistic to F. psychrophilum. By using multiple culture media, we obtained 174 isolates spanning 18 genera. Among them, Bosea sp. OX14 and Flavobacterium sp. GL7 respectively inhibited the growth of F. psychrophilum KU190628-78 and NCIMB 1947T, and produced antagonistic compounds of < 3 kDa in size. Sequences related to our isolates comprised 4.95% of skin microbial communities, and those related to strains OX14 and GL7 respectively comprised 1.60% and 0.17% of the skin microbiome. Comparisons with previously published microbiome data detected sequences related to strains OX14 and GL7 in skin of other rainbow trout and Atlantic salmon.