Abstract: Background: Culture of shiitake attracts attention of several researchers for its proved medicinal properties and antimicrobial effect and biodegradation abilities of phenolic compounds by producing large spectrum of enzymes. However, the growth kinetics of the mycelium is related to nutritional and environmental factors. A statistical approach according to the factorial design has been employed for identifying significant variables influencing biomass growth of Lentinula edodes Le119 strain and polyphenol removal from olive mill wastewater (OMW). Materials and Methods: The optimal formulation of substrates, including olive twigs and leaves (OTL), olive cake (OC) and OMW which acts as the wetting/moistening agent, for L. edodes culture was determined. Then, the growth factors including, initial pH, aeration rate, inoculums age, inoculums size, particle size and mineral solution were evaluated following the experimental screening design methodology using 2551//16 experiments. The fungal biomass was estimated by ergosterol technique and total phenol was determined according to the Folin-Ciocalteu assay. Results: The optimal formulation of substrate for L. edodes solid culture was OTL (20%)+OC (30%)+OMW (50%) allowing highest mycelial biomass production of 16.24 mg g–1 of substrate dry weight. Among the variables screened, the nitrogen added (through ammonium sulfate and urea) and spawn size had significant effects on mycelial biomass production as well as polyphenol removal from OMW. The optimized conditions of Le119 culture on solid olive substrate (SOS) with 20% of spawn and without nitrogen input led to mycelial biomass production of 48.72 mg g–1 of substrate dry weight and 82% drop in total phenols. Conclusion: The obtained results support the potential effective and profitable utilization of undiluted OMW in mixture with OTL and OC for L. edodes cultivation. However, farther experiments investigating the impact of these substrates on L. edodes fruiting yield and quality have to be conducted.