In today’s world, most of the currently used refrigerants in refrigeration systems have an important influence on global warming. This study presents the experimental and numerical consumption of thermal and energy performances comparison of new generation low GWP R290 and R1234yf refrigerants for a cold room. Evaporator temperatures of −9 °C, −6 °C, −3 °C, and condenser temperatures of 25 °C, 30 °C, 35 °C were utilized as the operational parameters. Results revealed that the highest COP values in the system for R290 and R1234yf were 3.99 and 3.41, respectively. An average 11.1% increase in the cooling capacity was determined with R290 compared to the R1234yf refrigerant. In the second part of the study, evaporator surface temperature and air velocity distributions in the cold room for R290 and R1234yf refrigerants occurring until the cold room temperature reached from the ambient temperature to 0 °C were numerically shown using Ansys Fluent 18.1. Numerical analysis using experimental data indicated that the evaporator surface had a lower temperature with R290 refrigerant than R1234yf. Results of the study highlighted the usability of R290 refrigerant instead of R1234yf in refrigeration systems in terms of thermodynamic performance.