Strawberry (Fragaria × ananassa Duch.) is one of the most widely cultivated fruit crop. Anthracnose caused by Colletotrichum spp. is a devastating disease of strawberry, causing large-scale strawberry losses worldwide. Chitinases act as defence proteins and are crucial for plant response to pathogens. Here, we isolated a class V Chitinase gene (designed as FnCHIT2, GenBank accession number MN709779) from Chinese wild diploid strawberry Fragaria nilgerrensis Schlecht (F. nilgerrensis), a species that exhibits high tolerance to anthracnose. Gene expression analysis showed that FnCHIT2 expression was highly induced after Colletotrichum gloeosporiodes inoculation and salicylic acid (SA) treatment. Subcellular localization analysis revealed the presence of FnCHIT2 in the plasma membrane. Recombinant FnCHIT2 protein was successfully expressed in E. coli Rosetta (DE3). Furthermore, we transformed FnCHIT2 into Col-0 wild type A. thaliana to perform functional analysis and evaluated the functions of Colletotrichum higginsianum and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). FnCHIT2 overexpression in A. thaliana showed enhanced resistance to C. higginsianum and Pst DC3000. Enhanced disease resistance of FnCHIT2 transgenic plants to C. higginsianum was correlated with pathogenesis-related gene 1 (AtPR1) and plant defensin 1.2 (AtPDF1.2) gene expression levels. These results provide evidence that FnCHIT2 may play an important role in response to fungal pathogens in strawberry. Our study provides an important theoretical reference for future strawberry resistance breeding.