Background Biotite is a program library for sequence and structural bioinformatics written for the Python programming language. It implements widely used computational methods into a consistent and accessible package. This allows for easy combination of various data analysis, modeling and simulation methods. Results This article presents major functionalities introduced into Biotite since its original publication. The fields of application are shown using concrete examples. We show that the computational performance of Biotite for bioinformatics tasks is comparable to individual, special purpose software systems specifically developed for the respective single task. Conclusions The results show that Biotite can be used as program library to either answer specific bioinformatics questions and simultaneously allow the user to write entire, self-contained software applications with sufficient performance for general application.