In this study, we aimed to examine the loss of strawberry fruit quality, including fruit firmness and number of microorganisms, after mechanical damage. Ripe greenhouse strawberries (Fragaria × ananassa ‘Tochiotome’) were subjected to drop-shock tests, categorized according to four different visual damage indexes, and stored for 7 days at either 5 or 15 °C. Fruit firmness and active microorganism proportion were investigated, and microorganism amplification and identification were performed. At 5 ℃, fruit firmness was effectively preserved until the end of the storage period, regardless of damage. The concentration of active microorganisms was determined based on adenosine triphosphate (ATP)-dependent luciferase activity and calculated as a relative value. Severely damaged fruit with effusion of juice stored at 15 °C showed the highest concentration of active microorganisms, compared to fruit stored at 5 °C, thus confirming the effectiveness of low-temperature storage for controlling microbial population size. According to the results of the amplification of the fungal 26 S ribosomal DNA and bacterial 16 S ribosomal RNA regions, and in agreement with the neighbor-joining consensus phylogram, the closest matches for bacterial and fungal sequences were Bacillus sp., Pseudomonas sp., and Curtobacterium sp., and Alternaria sp., Aspergillus sp., Cryptococcus sp., and Ustilago sp., respectively. The most abundant microorganisms from the fruit samples were Bacillus siamensis and Cryptococcus albidus. The reduction in fruit firmness during storage caused an increase in microbial concentration, especially in fruit stored at 15 °C, which may result in fruit softening and rapid microbial decay during storage. •Drop-shock test was used to simulate mechanical damage in strawberry fruit.•Concentration of microorganisms on strawberry skin increased with mechanical damage.•Negative correlation between fruit firmness and microbial concentration was found.•Greater softening and faster microbial decay occurred in fruit stored at 15 °C.•Effusion from injured tissues invited saprohite/pathogen invasion.