Hydroxy fatty acids have gained important attentions because of their special properties such as higher viscosity and reactivity compared to non-hydroxy fatty acids. The new bacterial isolate Pseudomonas aeruginosa (PR3) had been well studied to produce mono-, di-, and tri-hydroxy fatty acids from different unsaturated fatty acids. Of those hydroxy fatty acids, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced with high yield from oleic acid by P. aeruginosa PR3. Mostly, the substrates used for the production of hydroxy fatty acid by microorganisms were free fatty acids or synthetic lipids. However, it is highly plausible to use vegetable oils containing oleic acid such as olive oil for DOD production by P. aeruginosa PR3. As a first step to address the utilization of olive oil as a substrate for DOD production, we tried to determine the effect of vessel type on DOD production from olive oil by P. aeruginosa PR3. Of two different flask types of normal flask and baffled-flask, baffled-flask was highly effective for DOD production with all the media tested. Maximum increase of productivity by baffled-flask represented 221% with the medium supplemented with whey powder instead of lactose. Results from this study demonstrated that vessel type and medium composition could be both significant factors for DOD production from olive oil by P. aeruginosa PR3.