Prenylated phenolics are antimicrobials found in liquorice (Glycyrrhiza spp.). Liquorice spent is a by-product rich in prenylated phenolics obtained after water extraction of roots, and is currently not valorised. We analysed the prenylated phenolics composition of spent extracts from Glycyrrhiza glabra, G. inflata, and G. uralensis, their antimicrobial activity, cytotoxicity, and effects on Caco-2 cell viability. G. glabra, G. inflata, and G. uralensis spent extracts showed distinct phytochemical profiles. Antibacterial activity (Lactobacillus buchneri, Streptococcus mutans, and Staphylococcus aureus) of G. uralensis and G. inflata (MICs 25-250 μg mL-1) was higher than of G. glabra (MICs 75-1000 μg mL-1). Marker compounds glabridin, licochalcone A, and glycycoumarin were equally potent (MICs 12.5-25 μg mL-1). G. inflata and G. uralensis showed cytotoxicity at 500 μg mL-1, whereas G. glabra was not toxic up to 1000 μg mL-1, but showed reduced viability between 50-500 μg mL-1. Linking antibacterial activity of the liquorice spent extracts with cell viability showed that MICs against S. aureus coincide with concentrations where cell viability was not reduced, whereas for the other bacteria and yeasts MICs concurred at concentrations where cell viability was reduced. In this study we show that liquorice spent is a by-product rich in antibacterial prenylated phenolics that offers interesting oppurtunities for e.g. control of microorganisms and the discovery of novel plant-derived antimicrobials.