Phenolic compounds, both derivatives of benzoic and cinnammic acid, possess biologically valuable properties: anti-inflammatory, antioxidant, anticarcinogenic and others. Studies of the accumulation of these compounds focused mostly on plant material. Rich sources of these compounds are representatives of Basidiomycota taxon. The aim of the study was qualitative and quantitative HPLC analysis of phenolic acids in biomass from in vitro culture of selected edible mushroom species belonging to the phylum Basidiomycota: Agaricus bisporus, Boletus badius, Cantharellus cibarius. The investigations revealed the presence of the following acids: p-hydroxybenzoic, syringic and galic acid. Both the composition and the amount of phenolic acids in biomass of A. bisporus and Boletus badius were diverse. The total amount ranged from 6.07 mg·100 g−1 DW in A. bisporus to 14.78 mg·100 g−1 DW in Boletus badius. Syryngic acid amounts fluctuated in the range of 1.75–9.66 mg·100 g−1 DW, with its maximum in Boletus badius. Gallic acid dominated in the biomass of the same species (5.12 mg·100 g−1 DW). p-Hydroxybenzoic acid was found in biomass from in vitro culture of A. bisporus at levels 0.70 mg/100 g DW. In biomass of in vitro culture of Cantharellus cibarius no phenolic compounds were found before and after hydrolysis. The results of HPLC analyses show that in vitro culture of B. badius and A. bisporus are a good dietary source of phenolic compounds with antioxidant activity.