Understanding subcellular dynamic processes governing pathogenic mechanisms is a necessary step towards the development of new drugs and strategies against infectious diseases. Subcellular pathogenic mechanisms, such as viral invasion processes involve highly dynamic nanometric-scale objects and rapid molecular interactions that require the study of individual particle paths. Single-particle tracking methods allow visualizing and characterizing the dynamics of biological objects, and provide a straightforward and accurate means to understand subcellular processes. This review describes a number of particle-tracking methods in time-lapse microscopy sequences and provides examples of using such techniques to investigate mechanisms of host-pathogen interactions.