Rapid and accurate diagnosis of diseases is essential for early therapeutic intervention and efficacious treatment. Infectious diseases can be devastating with very high mortality and morbidity rates. These are caused by diverse organisms which replicate in their hosts. Enzymes are proteinaceous biocatalysts in nature, except ribozymes, which catalyze diverse reactions indispensable for life. Enzymes have numerous applications owing to their unique features like high substrate specificity, immense catalytic efficiency, diverse pH and temperature optima and the ability to be regulated. Enzymes play an essential and inimitable role in clinical diagnosis and management. Detection of enzymes produced by certain pathogens can help in apt and timely diagnosis of the infectious agent. This can favorably result in a positive outcome and successful therapeutic intervention and thus save lives. In this review, we attempt to summarize microbial enzymes including urease, coagulase, DNase, gelatinase, catalase, oxidase, βlactamase, tryptophanase and lipase, which are being used in the clinical diagnosis of diseases caused by pathogens and thus they positively influence the disease outcome. Owing to the various novel characteristics possessed by these enzymes, they have become a method of choice for rapid, accurate, economical and simple diagnostics.