As the microbial quality of fresh fruits and food by-products is relevant, in this study we focus on the development of the detection methods of strawberry phytopathogens: Colletotrichum sp., Phytophthora sp., Verticillium sp., and Botrytis cinerea. The primers were designed based on β-tubulin for Phytophthora sp., actin for Verticillium sp., and glyceraldehyde-3-phosphate dehydrogenase for Colletotrichum sp. and B. cinerea. The primers were used successively on pure microbial strains, as well as on naturally and artificially contaminated soil and strawberry plant samples. The detection limit for artificially contaminated soil was as follows: B. cinerea (100-10 000 spores g^-1 of soil), Colletotrichum sp. (1 000-100 000 spores g^-1 of soil), and Verticillium sp. (100 000 spores g^-1 of soil), whereas for artificially contaminated strawberry, it was the same for all tested pathogens (100 spores g^-1 of strawberry). The same primers were used to test bio-preparations, reducing the presence of these potential phytopathogens within different strawberry plant cultivars. The results demonstrated that a developed assay using specific primers designed based on functional genes can be used as a molecular alternative for monitoring and routine investigation of samples contaminated by four important fungal and fungal-like strawberry plant pathogens, including assessing environmental samples such as soil and plants (roots, shoots, fruits).