The value of fruits is determined by the quantity and variety of biologically active compounds they contain, and their benefits on human health. This work presents the first study of the biochemical composition and antibacterial activity of the new Japanese quince (JQ) cultivars ‘Darius’, ‘Rondo’, and ‘Rasa’ fruits. The total phenolic content (TPC) was determined using the Folin–Ciocalteu method and each compound was identified by HPLC High Performance Liquid Chromatography. The antimicrobial activity against three Gram-positive and three Gram-negative bacteria, and one yeast strain, was evaluated by the agar well diffusion method using three different concentrations. The free radical scavenging activity was determined using DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid) methods and ranged from 99.1 to 115.9 μmolTE/100 g, and from 372 to 682 μmolTE/100 g, respectively. TPC ranged from 3906 to 4550 mgGAE/100 g, and five compounds, isoquercitrin, rutin, (+)-catechin, (–)-epicatechin, and chlorogenic acid were identified. All JQ extracts possessed antimicrobial activity against Gram-positive and Gram-negative bacteria, and Enterococcus faecalis (ATCC 29212) was the most sensitive strain. These results indicate that JQ fruits are a significant source of bio-compounds, which can enrich the diet with strong antioxidants, and they are very promising as a substitute for chemical preservatives in the food and cosmetic industry.