The capacity of oyster mushroom (Pleurotus ostreatus) mycelium to produce meaty flavour compounds in the presence of amino acids and sugars was studied. The submerged liquid culture of P. ostreatus mycelium along with base medium made of defatted soybean powder, sucrose, potassium phosphate, and magnesium sulphate was incubated for 3 days at 25 °C. Cysteine, glutamine, or methionine and fructose, galactose, ribose, or xylose were added to the base medium to study the effect of amino acids and sugars on meaty flavour formation by trained panelists. The flavour compounds were isolated and identified by GC–MS and GC retention time of authentic compounds. The base medium required P. ostreatus, cysteine, ribose, and heat treatment to produce meaty flavour. The sulphur containing heterocyclic compounds such as 2,5-diethylthiophene, 2-formyl-5-methylthiophene, 3-ethyl-2-formylthiophene, and dimethylformyl thiophene were responsible for meaty flavour. These compounds were formed by non-enzymatic browning reaction between ribose and cysteine during heat treatment.