Glucosinolates (GSLs) and their breakdown products, isothiocyanates (ITCs), are bioactive compounds with anti-inflammatory, antioxidant, and anticancer properties, mediated through key pathways such as Nrf2, NF-κB, and epigenetic regulation. However, their limited and variable bioavailability remains a key challenge. This review summarises the current clinical evidence on GSLs and ITCs, with a focus on their health effects and metabolic fate in humans. Recent findings on enzymatic and microbial metabolism are discussed, along with results from interventions involving whole vegetables, sprouts, and extracts. Although promising effects on blood pressure, lipid profiles, and glycaemic control have been observed, clinical studies are often limited by small sample sizes, study heterogeneity, and high inter-individual variability, particularly related to gut microbiota and host metabolic phenotype. Challenges like inconsistent biomarkers, formulation variability, and tolerability issues complicate data interpretation. To realise their full potential, larger, standardised, microbiome-informed trials with validated biomarkers and optimised delivery are needed to clarify host–compound–microbiome interactions and support evidence-based disease prevention strategies.