Recently, the main aim of the road freight transportation sector has been the establishment of a cost-effective and sustainable operation because it is one of the most environmentally damaging and most expensive elements of the supply chains’ activities. The efficiency improvement and optimization of these transport activities can result in significant cost savings, which lead to increased competitiveness of the transport companies. Two new methods were elaborated for the optimization of road freight transport activities; therefore, this research is very innovative and up to date. The elaborated methods are as follows: (1) A new calculation method for the precise prime cost pre-calculation of transport tasks in order to determine an accurate transport fee, thus ensuring the company’s profit; furthermore, the losses can be eliminated in order to provide competitiveness to the transport company. (2) A new optimization method for the refueling procedure of international transport trips in order to minimize the total fuel cost of the transport trips taking into consideration the different unit fuel prices at the different stations. Therefore, the elaborated optimization method on the one hand helps in selecting the optimal petrol station and, on the other hand, defines the optimal amount of fuel to be refueled. Based on the newly developed methods, two decision-supporting software applications were developed to establish more profitable and sustainable transportation. The added value of the developed calculation methods and software applications is that, recently, both the prime cost calculation and the fuel supply optimization have not been supported by software. This is the reason the two developed methods and two software applications are innovative and unique. The newly developed software applications were successfully implemented at transport companies. The correctness of both elaborated mathematical methods was validated using the developed software in real case studies.