China is the largest strawberry producer and exporter worldwide and has been constantly challenged by fruit rot disease in recent years. Symptoms of various diseases on strawberry fruit were observed in Huangqiyuan Base, an important producing region in Shandong province, and symptomatic samples were collected from January to April 2021 for follow-up studies. In the present study, 137 isolates were obtained and classified into nine species based on morphological characteristics and multi-locus phylogenetic analysis (ITS, GAPDH, HIS3, RPB2, EF-1α, HSP60, G3PDH, or/and TUB2), including Botrytis cinerea, B. fabiopsis, Alternaria alternata, A. tenuissima, Fusarium proliferatum, F. graminearum, F. ipomoeae, F. incarnatum, and Colletotrichum siamense. Pathogenicity results suggested that all nine pathogenic species could induce fruit to exhibit symptoms similar to those naturally infected in fields. The symptoms around the inoculation points were various, including densely white mycelia caused by Botrytis species, fading and depression caused by Fusarium, black-brown rot caused by Alternaria, as well as shrinkage and dehydration caused by Colletotrichum. Overall, B. cinerea was the dominant pathogen, accounting for 61.3% of the total isolates, and showed significantly higher virulence against strawberry fruit than other species. In addition, this is the first report to identify B. fabiopsis, A. alternata, A. tenuissima, F. proliferatum, F. graminearum, F. ipomoeae, and F. incarnatum as causal agents of strawberry fruit rot in Shandong province, China.