Increasingly, neuroimaging researchers are exploring the use of real-time functional magnetic resonance imaging (rt-fMRI) as a way to access a participant’s ongoing brain function throughout a scan. This approach presents novel and exciting experimental applications ranging from monitoring data quality in real time, to delivering neurofeedback from a region of interest, to dynamically controlling experimental flow, or interfacing with remote devices. Yet, for those interested in adopting this method, the existing software options are few and limited in application. This presents a barrier for new users, as well as hinders existing users from refining techniques and methods. Here we introduce a free, open-source rt-fMRI package, the Pyneal toolkit, designed to address this limitation. The Pyneal toolkit is python-based software that offers a flexible and user friendly framework for rt-fMRI, is compatible with all three major scanner manufacturers (GE, Siemens, Phillips), and, critically, allows fully customized analysis pipelines. In this article, we provide a detailed overview of the architecture, describe how to set up and run the Pyneal toolkit during an experimental session, offer tutorials with scan data that demonstrate how data flows through the Pyneal toolkit with example analyses, and highlight the advantages that the Pyneal toolkit offers to the neuroimaging community.