Nitroreductases, enzymes found mostly in bacteria and also in few eukaryotes, use nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor for their activity and metabolize an enormous list of a diverse nitro group-containing compounds. Nitroreductases that are capable of metabolizing nitroaromatic and nitro heterocyclic compounds have drawn great attention in recent years owing to their biotechnological, biomedical, environmental, and human impact. These enzymes attracted medicinal chemists and pharmacologists because of their prodrug selectivity for activation/reduction of nitro compounds that wipe out pathogens/cancer cells, leaving the host/normal cells unharmed. It is applied in diverse fields of study like prodrug activation in treating cancer and leishmaniasis, designing fluorescent probes for hypoxia detection, cell imaging, ablation of specific cell types, biodegradation of nitro-pollutants, and interpretation of mutagenicity of nitro compounds. Keeping in view the immense prospects of these enzymes and a large number of research contributions in this area, the present review encompasses the enzymatic reaction mechanism, their role in antibiotic resistance, hypoxia sensing, cell imaging, cancer therapy, reduction of recalcitrant nitro chemicals, enzyme variants, and their specificity to substrates, reaction products, and their applications.