This study was performed to investigate the effect of high hydrostatic pressure (HHP) on the conversion of linoleic acid, conjugated linoleic acid (CLA), and α-linolenic acid (α-LNA) as substrates by Bifidobacterium breve LMC520 and to optimize the HHP condition. Cell mixture were tested under HHP in a variety of conditions such as temperature, time, pressure, and pre- or post-treatment with substrates. The cis-9,trans-11 CLA producing activity of B. breve LMC520 was increased by HHP, whereas trans-9,trans-11 CLA producing activity was decreased. Optimal HHP conditions for the highest CLA production were obtained at 100 MPa for 12 h at 37 °C. Post-treatment groups showed higher conversion activity of substrates than pretreatment groups. Post-treatment groups decreased trans-9,trans-11 CLA and other CLnA, whereas the pretreatment groups increased them. It is concluded that HHP treatment could be an important factor to enhance CLA and CLnA production and for reducing trans-fatty acids.