Direct numerical simulations (DNS) are conducted of a model hydrocarbon–nitrogen mixing layer under supercritical conditions. The temporally developing mixing layer configuration is studied using heptane and nitrogen supercritical fluid streams at a pressure of 60 atm as a model system related to practical hydrocarbon-fuel/air systems. An entirely self-consistent cubic Peng–Robinson equation of state is used to describe all thermodynamic mixture variables, including the pressure, internal energy, enthalpy, heat capacity, and speed of sound along with additional terms associated with the generalized heat and mass transport vectors. The Peng–Robinson formulation is based on pure-species reference states accurate to better than 1% relative error through comparisons with highly accurate state equations over the range of variables used in this study (600 [les ] T [les ] 1100 K, 40 [les ] p [les ] 80 atm) and is augmented by an accurate curve fit to the internal energy so as not to require iterative solutions. The DNS results of two-dimensional and three-dimensional layers elucidate the unique thermodynamic and mixing features associated with supercritical conditions. Departures from the perfect gas and ideal mixture conditions are quantified by the compression factor and by the mass diffusion factor, both of which show reductions from the unity value. It is found that the qualitative aspects of the mixing layer may be different according to the specification of the thermal diffusion factors whose value is generally unknown, and the reason for this difference is identified by examining the second-order statistics: the constant Bearman–Kirkwood (BK) thermal diffusion factor excites fluctuations that the constant Irwing–Kirkwood (IK) one does not, and thus enhances overall mixing. Combined with the effect of the mass diffusion factor, constant positive large BK thermal diffusion factors retard diffusional mixing, whereas constant moderate IK factors tend to promote diffusional mixing. Constant positive BK thermal diffusion factors also tend to maintain density gradients, with resulting greater shear and vorticity. These conclusions about IK and BK thermal diffusion factors are species-pair dependent, and therefore are not necessarily universal. Increasing the temperature of the lower stream to approach that of the higher stream results in increased layer growth as measured by the momentum thickness. The three-dimensional mixing layer exhibits slow formation of turbulent small scales, and transition to turbulence does not occur even for a relatively long non-dimensional time when compared to a previous, atmospheric conditions study. The primary reason for this delay is the initial density stratification of the flow, while the formation of strong density gradient regions both in the braid and between-the-braid planes may constitute a secondary reason for the hindering of transition through damping of emerging turbulent eddies.