Single-molecule detection and analysis of organic fluorescent molecules and proteins are presented, with emphasis on the underlying principles, methodology and the application of single-molecule analysis at room temperature. This Minireview is mainly focused on the application of confocal and near-field optical microscopy to investigate the photodynamics of individual molecules embedded in ultrathin polymer layers. We discuss rotational mobility of individual probe molecules in polystyrene and poly(methylmethacrylate) thin films, fluorescence lifetime trajectories and their spatial distribution, and real-time singlet-triplet dynamics. As a whole, the single-molecule photodynamics observed is due to the dynamic nature of both polymers at room temperature, where local polymer conformational dynamics modulates the oxygen concentration and diffusion on a molecular scale, influencing the fluorescence lifetime and intersystem crossing parameters. We also discuss the photodynamics of individual autofluorescent proteins, in particular the on/off blinking and the apparent stability of the protein against bleaching. These studies illustrate the unique information obtainable with the single-molecule approach, information that is otherwise hidden in ensemble-averaged measurements.