There is an immediate requirement for the novel antimicrobials in aquaculture, mainly because of the high rate of evolving antibiotic-resistant bacteria. Screening compounds for antimicrobial activity against fish pathogenic bacteria is a cost-effective and sustainable solution to address this challenge. In the present work,we investigated the in vitro antibacterial activity of 7 synthetic antimicrobial peptides (AMPs) against 12 clinical isolates of pathogenic bacteria, and two reference strains, Aeromonas hydrophila (ATCC 7966), and Escherichia coli ATCC 25922. The clinical isolates were previously recovered from the internal organs of 4 species of freshwater fish (Colossoma macropomum, Oreochromis niloticus, Pseudoplatystoma fasciatum, and Pterophyllum scalare) positively diagnosed with a bacterial infection, sampled during disease outbreaks at six fish farms or facilities located in South, and Southeast Brazil. AMPs demonstrated potent antibacterial activity against A. jandaei (Kr12a5), C. freundii (Kr12a5; [Trp3,5,10] des-Cys11, Lys12, Lys13-(pBthTX-I)2K; des-Cys11, Lys12, Lys13-(pBthTX-I)2K; LL 37; and Hylin a1), S. agalactiae (Hylin a1; [Trp3,5,10] des-Cys11, Lys12, Lys13-(pBthTX-I)2K; and LL 37), L. garviae (Hylin a1; and [Trp3,5,10] des-Cys11, Lys12, Lys13-(pBthTX-I)2K), and V. fluvialis (Kr12a5). These findings suggest the potential of some AMPs as a treatment option for bacterial diseases in aquaculture.