Conjugated linoleic acid (CLA) is a group of positional and geometric isomers widely admired for its anti-inflammatory and anti-carcinogenic effects. Enzymes responsible for bioconverting linoleic acid (LA) into CLA are diverse, found in a wide range of microorganisms from lactic acid bacteria (LAB) to non-lactic acid bacteria (non-LAB). Various LAB and non-LAB strains show promising results in CLA production. Enzymes such as linoleate isomerase (LAI), Myosin cross-reactive antigen (MCRA), and oleate hydratase, involved in CLA production, are also diverse in nature. They originate from various sources and have demonstrated potential to biohydrogenate LA and other similar substrates. Comparative analysis of substrate–enzyme dynamics revealed substantial structural conservation alongside functional divergence among various biohyrogenating enzyme-associated pathways, suggesting evolutionary adaptation toward fatty acid detoxification and membrane homeostasis. Furthermore, environmental and physiological factors such as substrate availability, oxygenation, pH, temperature, and growth phase were found to significantly influence CLA biosynthesis. Despite considerable advances, several mechanistic aspects of enzyme cooperation, pathway regulation, and evolutionary relationships remain unresolved. This article critically examines various aspects of substrate–enzyme dynamics, their ecology, evolution, and molecular regulation. It aims to bridge the knowledge gap in catalytic mechanisms and regulation while summarizing available information on various enzymes responsible for the bioconversion of LA to CLA, implicating fundamental microbiology and biotechnology.