The biocatalysis of mushroom tyrosinase was optimized in chloroform medium using as substrates selected phenolic compounds, including chlorogenic acid (CA), p-aminophenol (pAP) and hydroquinone (HQ). The specific activity of tyrosinase was found to be much higher in the chloroform medium than that obtained in the aqueous one. The optimal pH and temperature as well as other kinetic parameters for tyrosinase biocatalysis was also determined. The presence of catechol in the chloroform medium resulted in an increase in tyrosinase activity when CA was used as substrate; however, no activatory effect was found with pAP or HQ. The presence of ethylenediamine tetraacetic acid in the chloroform medium showed different effects on tyrosinase activity depending on the nature of the substrate. FT-IR analyses confirmed the bioconversion of selected phenolic substrates into o-quinones by tyrosinase activity in the chloroform medium.