Antioxidants are decisive bio molecules that alleviate the damaging effects of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are incessantly formed as by-products of usual cellular metabolism. Organisms across all domains of life including plants, animals and microbes have evolved complex antioxidant systems to uphold redox homeostasis and guarantee survival under both normal and stress conditions. The metabolism of antioxidants comprises both enzymatic and non-enzymatic components. Enzymatic antioxidants comprise superoxide dismutase, catalase, glutathione peroxidase (GPx) and peroxiredoxins, which act synergistically to counteract various ROS. Non-enzymatic antioxidants including ascorbic acid, tocopherols, glutathione, flavonoids, carotenoids and polyphenols, function by directly scavenging free radicals or by regenerating oxidized antioxidant molecules. In plants, antioxidant systems are highly diversified in their components such as mitochondria, chloroplasts and peroxisomes, and plants undergo oxidative stress through various mechanisms including drought, salinity, photosynthesis and biotic and abiotic stress. All these mechanisms were controlled by the network of antioxidants and protect plant tissues. In the case of animals, the antioxidants help in oxidative damage through metabolic activity, ageing and some diseases that involve the Nrf2-ARE signalling pathway and it maintains the gene coding, etc. Similarly, many microorganisms like bacteria, fungi and archaea comprise antioxidant mechanisms in the case of host–pathogen interactions, and exposure to environmental conditions by synthesizing metabolites like mycothiol, ergothioneine and microbial carotenoids. In this chapter, the antioxidant metabolism and mechanisms in different organisms including plants, animals and microbes were discussed and provided deep insights into the applications in medicine, agriculture and industry which help in future to carry synthetic biology by enabling the enhanced antioxidant traits in probiotics, crops and therapeutics and helps in improving health-related issues.