Amoebic gill disease in teleost fish is caused by the marine parasite Neoparamoeba perurans. To date, the role of antimicrobial peptides β-defensins and cathelicidins in this infection have not been explored. Using a high-throughput microfluidics quantitative polymerase chain reaction system (Biomark HD™ by Fluidigm), this study aimed to: firstly, to investigate organ-specific expression of antimicrobial peptide genes β-defensin-1, -3 and -4 and cathelicidin 2 in healthy Atlantic salmon; secondly, to compare the expression of these antimicrobial peptide genes in healthy versus asymptomatic Atlantic salmon seven days post-challenge with Neoparamoeba perurans. Results from this study indicate expression of the β-defensin and cathelicidin genes in the selected organs from healthy Atlantic salmon. Furthermore, a statistically significant upregulation of β-defensins -3 and -4 and cathelicidin 2 was detected in gill of parasite-challenged salmon. The upregulated cathelicidin and β-defensin genes in gill could indicate novel potential roles in innate immune responses to Neoparamoeba perurans.