Single-particle tracking (SPT) is a key tool for quantitative analysis of dynamic biological processes and has provided unprecedented insights into a wide range of systems such as receptor localization, enzyme propulsion, bacteria motility, and drug nanocarrier delivery. The inherently complex diffusion in such biological systems can vary drastically both in time and across systems, consequently imposing considerable analytical challenges, and currently requires an a priori knowledge of the system. Here we introduce a method for SPT data analysis, processing, and classification, which we term “diffusional fingerprinting.” This method allows for dissecting the features that underlie diffusional behavior and establishing molecular identity, regardless of the underlying diffusion type. The method operates by isolating 17 descriptive features for each observed motion trajectory and generating a diffusional map of all features for each type of particle. Precise classification of the diffusing particle identity is then obtained by training a simple logistic regression model. A linear discriminant analysis generates a feature ranking that outputs the main differences among diffusional features, providing key mechanistic insights. Fingerprinting operates by both training on and predicting experimental data, without the need for pretraining on simulated data. We found this approach to work across a wide range of simulated and experimentally diverse systems, such as tracked lipases on fat substrates, transcription factors diffusing in cells, and nanoparticles diffusing in mucus. This flexibility ultimately supports diffusional fingerprinting’s utility as a universal paradigm for SPT diffusional analysis and prediction.