The fluorescence decays (FDs) of 27 dried vegetative bacteria, bacterial endospores, fungi, and pollens were measured and determined using a stroboscopic technique. Pulsed nanosecond LED sources, emitting light at wavelengths of 280, 340, and 460 nm, were used for the excitation of biological samples. The implicit advantages of the stroboscopic method are high sensitivity, speed of a single measurement (10–60 s), miniaturization of the device, and relatively low price compared to the typical lifetime methods. The Principal Component Analysis (PCA) method was used for chemometric analysis. It was found that the excitation at 340, 460, and data merged from 340 and 460 nm effectively separate individual groups of biological substances. These findings provide evidence that fluorescence decay data may allow the classification of the biological samples, and the FDs measurement method can be complementary to the study of fluorescence spectra.