Dataset underlying the following paper: Courtecuisse, E.; Marchetti, E.; Oxborough, K.; Hunter, P.D.; Spyrakos, E.; Tilstone, G.H.; Simis, S.G.H. Optimising Multispectral Active Fluorescence to Distinguish the Photosynthetic Variability of Cyanobacteria and Algae. Sensors 2023, 23 This study assesses the ability of a new active fluorometer, the LabSTAF, to diagnostically assess the physiology of freshwater cyanobacteria in a reservoir exhibiting annual blooms. Specifically, we analyse the correlation of relative cyanobacteria abundance with photosynthetic parameters derived from fluorescence light curves (FLCs) obtained using several combinations of excitation wavebands, photosystem II (PSII) excitation spectra and the emission ratio of 730 over 685 nm (Fo(730/685)) using obtained with excitation protocols with varying degrees of sensitivity to cyanobacteria and algae. FLCs captured obtained with blue excitation (B) and green–orange–red (GOR) excitation wavebands capture physiology parameters of algae and cyanobacteria, respectively. The green–orange (GO) protocol, expected to have the best diagnostic properties for cyanobacteria, did not guarantee PSII saturation. PSII excitation spectra showed distinct response from cyanobacteria and algae, depending on spectral optimisation of the light dose. Fo(730/685), obtained using a combination of GOR excitation wavebands, Fo(GOR, 730/685), showed a significant correlation with the relative abundance of cyanobacteria (linear regression, p-value < 0.01, adjusted R2 = 0.42). We recommend using, in parallel, Fo(GOR, 730/685), PSII excitation spectra (appropriately optimised for cyanobacteria versus algae), and physiological parameters derived from the FLCs obtained with GOR and B protocols to assess the physiology of cyanobacteria and to ultimately predict their growth. Higher intensity LEDs (G and O) should be considered to reach PSII saturation to further increase diagnostic sensitivity to the cyanobacteria component of the community.