For the first time, a soil bacterium, designated Pseudomonas aeruginosa, was isolated based on its ability to grow on tyrosol as a sole source of carbon and energy. During growth on tyrosol, this strain was capable of promoting the formation of a significant amount of hydroxytyrosol and trace quantities of parahydroxyphenyl acetic acid and 3,4-dihydroxyphenyl acetic acid. The products were confirmed by high-performance liquid chromatography and gas chromatography-mass spectrometry analyses. Using an optimized tyrosol concentration of 2 g liter−1, the maximal hydroxytyrosol yield (80%) was achieved after a 7-h reaction in a growth experiment. To enhance the formation of hydroxytyrosol and prevent its degradation, a resting-cell method using P. aeruginosa was performed. The growth state of the culture utilized for biomass production, the carbon source on which the biomass was grown, the concentration of the biomass, and the amount of tyrosol that was treated were optimized. The optimal yield of hydroxytyrosol (96%) was obtained after a 7-h reaction using 4 g of tyrosol liter−1 and 5 g of cells liter−1 pregrown on tyrosol and harvested at the end of the exponential phase. This proposed procedure is an alternative approach to obtain hydroxytyrosol in an environmentally friendly way. In addition, the reaction is easy to perform and can be adapted to a bioreactor for industrial purposes.