In the course of searching for novel natural antioxidants from Eucalyptus globulus leaf waxes, 4-hydroxytritriacontane-16,18-dione was newly isolated and identified alongside the known 16-hydroxy-β-tritriacontanone. This new compound showed strong antioxidative activity in water/alcohol systems (measured by thiocyanate and TBA methods) but no activity in oil systems, and diketones with long alkyl side chains exhibited stronger activity than other β-diketone analogues. Given the growing focus on natural antioxidants (preferred over synthetic ones like BHA and BHT for food safety) and the limitations of tocopherols (less effective when used alone and high in cost), this study aimed to isolate new types of antioxidants from natural resources, particularly Eucalyptus leaf waxes (hypothesized to protect endogenous essential oils from oxidative degradation). Previous work had identified the novel β-diketone antioxidant n-tritriacontane-16,18-dione in E. globulus leaf wax and suggested other minor antioxidative substances. This paper reports the isolation and structural elucidation of two new β-diketone analogues (S-2A and S-2B) and their antioxidative activities in model systems, as well as the investigation of structure-activity relationships for β-diketone derivatives. Spectroscopic analyses (UV, IR, NMR, MS) determined S-2A as 16-hydroxy-18-tritriacontane and S-2B as 4-hydroxytritriacontane-16,18-dione. Antioxidative assays showed that S-2B had marked activity in water/alcohol systems, comparable to n-tritriacontane-16,18-dione, while S-2A exhibited only weak activity; none of the leaf wax components showed activity in oil systems. Structure-activity relationship studies revealed that long hydrocarbon side chains on both sides of the β-diketone moiety are essential for antioxidative activity, as shorter or single-side-chain analogues (e.g., acetyl acetone, syncarpic acid, n-duodecoylacetone) had no activity. The β-diketone moiety itself is critical for activity in aqueous systems. Preliminary tests showed Eucalyptus β-diketones have strong activity in mayonnaise, with ongoing experiments on other food systems (e.g., salad dressing, frozen meat/fish). Additionally, leaf waxes from other plant species (Acacia, Rhododendron) also demonstrated strong antioxidative activity, likely due to long-chain β-diketones.