Strawberry (Fragaria x ananassa Duch.) is a widely grown vegetable crop in Egypt. During a routine survey,wilt and root rot symptoms were observed on strawberry plants cv. Festival. Samples were collected and isolation was done on potato dextrose agar (PDA) medium. The frequently isolated fungi were Pestalotiopsis spp. along with other associated fungi. Koch's postulates were fulfilled against two strawberry cultivars Fortuna and Festival under greenhouse conditions. Pestalotiopsis fungal isolates were identified by amplification and sequencing of ITS, TEF-1α and β-tubulin gene regions. The BLASTn search of GenBank database revealed that all obtained sequences exhibited similarity of 99–100% to Neopestalotiopsis rosae. Four fungicides were evaluated against N. rosae through in vitro and in vivo trials. In vitro trials revealed that fungicides Thiram and Hymexazole inhibited the mycelial growth of N. rosae at concentrations 1000 and 1250 ppm, respectively. In vivo trials showed that Thiram reduced significantly disease severity of crown and root rot on both cultivars.