This updated and extended second edition of the textbook introduces the basic concepts of bioinformatics and enhances students' skills in the use of software and tools relevant to microbiology research. It discusses the most relevant methods for analysing data and teaches readers how to draw valid conclusions from the observations obtained. Free software and servers available on the Internet are presented in an updated version of 2023 and more advanced stand-alone software is proposed as a second option. In addition, new tools for microbial genome analysis and new flowcharts that complement the didactic elements have been added. Exercises and training questionnaires are included at the end of each chapter to facilitate learning.  The book is aimed at Ph.D. students and advanced undergraduate students in microbiology, biotechnology, and (veterinary) medicine with little or basic knowledge of bioinformatics.