BackgroundBiotite 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.ResultsThis 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.ConclusionsThe 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.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12859-023-05345-6.