Onion (Allium cepa L.) contains various bioactive organosulfur compounds, including recently identified species with a 3,4-dimethylthiolane ring structure. In this study, the biological activities of 24 onion-derived thiolane samples were comprehensively evaluated, focusing on antioxidant, anti-inflammatory, antidiabetic, and antimicrobial potential. Tested thiolanes showed significant antioxidant activity by reducing intracellular reactive oxygen species (ROS) levels in THP1-Blue™ NF-κB cells; however, some tested derivatives exhibited mild pro-oxidant effects. In assays targeting metabolic regulation, several compounds showed partial activation of the peroxisome proliferator-activated receptor gamma (PPARγ) pathway, particularly allithiolane B isomers, achieving 22–26% of the maximal response induced by the known full agonist rosiglitazone, suggesting potential as PPARγ modulators. Anti-inflammatory assessments revealed minimal direct inhibition of NF-κB signaling, except for onionin A, which modestly attenuated NF-κB activity. Cytotoxic evaluation indicated safety at physiological concentrations (up to 10 μM), though some thiolanes significantly reduced cell viability at higher concentrations (50 μM), highlighting their potential for selective cytostatic applications. Antimicrobial screening indicated limited activity against bacteria and fungi at tested concentrations, except for moderate antifungal effectiveness against Candida albicans shown by allithiolane D and cepathiolactols F. Overall, these results emphasise that onion thiolanes are multifunctional molecules capable of modulating oxidative stress, metabolic regulation and inflammation, supporting their potential use in nutraceutical strategies aimed at chronic disease prevention and management.