Fruit quality is strongly affected by genotype and harvest date. In this study, parameters regarding fruit quality, bioactive compounds, and antioxidant capacity of different strawberry cultivars at three harvesting dates were quantified to elucidate the influence of genotype and harvest date on strawberry quality. The results showed that harvest date was the major contributor to appearance color, TSS, TA, and TSS/TA ratio of strawberries, while genotype mainly affected firmness, anthocyanin content, and antioxidant capacity. Moreover, the interaction of genotype and harvest date had a primary influence on the content of ascorbic acid. The content of total phenolics and amino acids received the similar influence caused by genotype and harvest date. However, the interaction of genotype and harvest date significantly affected total phenolic content as well. These findings give a better understanding of the influence of the genotype and harvest date on strawberry, which might contribute to breed cultivars with more attractive fruits in terms of quality acceptance and nutritional value.