Energy serves as an essential factor in a nation’s socio-economic progress. With global industrialisation on the rise and the world population expanding, there is an escalating need for a consistent and reliable energy supply to fuel rapid economic growth. The volatile nature of oil market prices, depleting oil reserves, and the adverse impact of greenhouse gas emissions led to global warming and other challenges associated with using fossil fuels. One of the finest alternative energy sources is biohydrogen, a clean, nonpetroleum-based, carbon-free fuel that produces only water when combusted without releasing greenhouse gases. Microbiology is fundamental to biohydrogen production, as it harnesses the metabolic capabilities of various microbes to convert organic matter into hydrogen gas. Microbes oxidise the organic substrates to produce electrons, which are transferred to free protons to form molecular hydrogen with hydrogenases, a reversible reaction. Understanding the metabolic pathways and the enzymes of photo-fermentative and anaerobic microbes like nitrogenases and hydrogenases is crucial. Nitrogenase can reduce nitrogen to ammonia and generate hydrogen as a by-product under anaerobic and nitrogen-deficient conditions. The ongoing study of enzymology in biohydrogen production is essential for developing strategies to enhance enzyme activity, stability, and metabolic pathways. The future scope of biohydrogen production is vast and interdisciplinary, requiring microbiology, engineering, and economic advancements. Overcoming present challenges will pave the way for biohydrogen to become a key component of a sustainable energy future.