This paper addresses the optimal working fluid selection for organic Rankine cycle recovering heat from heavy-duty internal combustion engines. Four cases are considered featuring two different engine exhaust temperatures (245 °C vs 354 °C) and two scenarios (maximum recovery of mechanical power vs. cogeneration of low-temperature heat). The analysis includes both pure fluids, including recently synthesized refrigerants, and binary mixtures. To perform a fair comparison between the different fluids, a computationally efficient cycle optimization approach, able to determine the maximum achievable efficiency for each working fluid, is adopted. The approach combines the evolutionary optimization algorithm PGS-COM with a rigorous heat integration methodology. The most efficient fluids are HCFO-1233zde, HFE-245fa2, HFO-1336mzz, HFE-347mcc, HFE-245cb2 and Novec 649 for the engine with lower temperature exhausts (reaching an ORC mechanical efficiency of 18.6–19.9%), and cyclopentane, ammonia, HCFO-1233zde, HFE-245fa2, HFO-1366mzz for the engine with higher temperature (reaching 23.76–22.70% efficiency). Compared to pure fluids, the use of optimized binary mixtures does not appear to lead a considerable efficiency gain.