We present results from a study evaluating the utility of supervised machine learning to classify single particle ultraviolet laser-induced fluorescence (UV-LIF) signatures to investigate airborne primary biological aerosol particle (PBAP) concentrations in a busy, multifunctional building using a Multiparameter Bioaerosol Spectrometer. First we introduce and demonstrate a gradient boosting ensemble decision tree algorithm’s ability to accurately classify laboratory generated PBAP samples into broad taxonomic classes with a high level of accuracy. We then develop a framework to appraise the classification accuracy and performance using the Hellinger distance metric to compare product parameter probability density function similarity; this framework showed that key training classes were sufficiently different in terms of particle fluorescence and morphology to facilitate classification. We also demonstrate the utility of including advanced morphological parameters to minimise inter-class conflation and improve classification confidence, where relying on the fluorescent spectra alone would likely result in misattribution. Finally, we apply these methods to ambient data collected within a large multi-functional building where ambient bacterial- and fungal-like classes were identified to display trends corresponding to human activity; fungal-like classes displayed a consistent diurnal trend with a maximum at midday and hourly peaks correlating to movements within the building; bacteria-like aerosol displayed complex, episodic events during opening hours. All PBAP classes fell to low baseline concentrations when the building was unoccupied overnight and at weekends.