A fluorescence quenching method was used to determine the equilibrium binding constants for the association of acridine, acridine orange, and acridine yellow G to humic acid. The fluorescence of each polycyclic aromatic nitrogen heterocycle (PANH) was monitored as aliquots of humic acid were added, and a Stern-Volmer plot was produced in which the slope is the equilibrium constant of the binding reaction. The quenching experiments were performed at temperatures of 30, 35, 40, and 45 °C. A van't Hoff plot generated from the equilibrium binding constants as a function of temperature for a given PANH resulted in a linear plot. Calculation of the ΔHbinding, ΔGbinding, and ΔSbinding for each PANH leads to the conclusion that the equilibrium binding constant, and ΔGbinding, may be predictors of bioavailability. The other thermodynamic quantities, ΔHbinding and ΔSbinding, are helpful in understanding the relative binding of the compounds. For example, acridine yellow G appears to be the least bioavailable of the three PANHs studied because of its strong ΔHbinding = -29.8 kJ/mol, which leads to ΔGbinding = -0.71 kJ/mol. While acridine orange and acridine have similar ΔHbinding values, acridine orange is more likely to bind to humic acid because the ΔSbinding for the process is less negative. Thermodynamic values and equilibrium binding constants for all three compounds are reported.