This paper presents the effects of the physiological reaction of the main cool-season grass species used for turf in a temperate climate: perennial ryegrass, Kentucky bluegrass, and tall fescue, on a twice-a-year trinexapac-ethyl (TE) application in late spring and early autumn, seasons of rapid turf growth. The fully established turf plots in the split-plot system of three replicates, with three cultivars/species, were treated by TE (1.5 and 4.5 g/100 m2). The 4.5 g/100 m2 was harmful to Kentucky bluegrass. The perennial ryegrass responded by dose-dependent growth inhibition; 30–60% in spring and 25–40% in autumn for lower and higher doses, respectively. Tall fescue responded by 50% growth inhibition independently of concentration and season. Plant physiological responses, visualized as graphs of fluorescence data, revealed the stress of Kentucky bluegrass upon high TE dose. Based on principal component analysis (PCA) analysis, three groups were distinguished: perennial ryegrass varieties from high and low TE treatments and Kentucky bluegrass varieties from high TE. TE-dependent growth reduction with no significant quality decrease benefits the environment by reducing carbon footprint machine operations (mowing). Utilizing fluorescence measurement may help to manage turf physiology.