Representatives of the Basidiomycota (Basidiomycetes) are an important source of biologically active compounds. In their fruiting bodies compounds of different actions were detected e.g. antiviral, bacteriostatic, fungistatic, antiparasitic, anticancer, anti-inflammatory and also hipoglicemic. Often, there are considerable difficulties in obtaining their specimens from the natural state. For this reason, more and more studies are undertaken to study chemical composition not only specimens obtained from natural state but also extracted from mycelial in vitro cultures. Many studies demonstrated qualitative and quantitative differences between fruiting bodies and mycelial in vitro cultures in the production of certain chemical groups. The paper describes mycelial cultures methodology (i.a. constitution, optimization of their conditions, the types of substrates used in in vitro cultures of higher fungi and finally types of cultures). Moreover, there are characterized the most important biologically active compounds produced by the Basidiomycota mycelium in vitro cultures.