Microbiology is an area with a high research production, classified in different branches. Thus, it is difficult to identify research trends without many backgrounds. The present paper aims to apply bibliometric methods to identify the highly cited papers that contribute to the Microbiology development. Furthermore, a science mapping analysis allows discovering the main thematic areas covered by those detected papers. From the 572 153 papers (articles and reviews) indexed in the Web of Science Microbiology category (1900-2017), a total of 645 highly cited papers were detected. Concerning the main scientific producers in Microbiology, Applied and Environmental Microbiology as a journal, R. Knight from the University of Colorado (USA) as an author and the USA as a country are the most productive actors. Nevertheless, taking into account the gross domestic product per capita, China and Spain are remarkable countries. Furthermore, according to the relative priority index (RPI), Denmark, Belgium, Spain and The Netherlands are the countries that make a higher scientific effort in this field. About the co-word analysis, several important themes were detected, such as Next Generation Sequencing, Gene, Escherichia Coli or Gene Expression. Concerning these results, this work supposes a framework on which to base future research.