Viruses are responsible for more than 50% of annual potato tuber yield losses and cause great economic damage. The traditional Croatian potato cultivar ‘Brinjak’ is important for local growers because of its economically profitable production and as a gene pool for future breeding programs. However, the full genetic potential of the cultivar cannot be exploited due to virus infection. In this study, we attempted to eliminate potato virus M (PVM) and potato virus S (PVS) from potato cultivar ‘Brinjak’ and to evaluate the effects on physiological parameters and yield. Shoot apices were isolated from PVM + PVS-infected sprouts and cultivated for six weeks on MS medium with the addition of 50 or 100 mg L−1 ribavirin. The surviving shoot apices were micropropagated. The in vitro post-eradication period lasted 200 days. DAS-ELISA and RT-PCR were performed on R0 and R1 plants 90 days after acclimatization to determine the sanitary status of the plants. Chlorophyll fluorescence and multispectral imaging were performed on the R0 plants at the same time. The success of PVS elimination was 33% at both ribavirin concentrations. However, neither concentration was successful in eliminating PVM. Plants with mixed infection (PVM + PVS) had more severe disease symptoms compared to PVM-infected plants, affecting photochemistry and multispectral parameters and, consequently, yield. PVM + PVS plants had significantly lower number and weight of tubers per plant and lower average tuber weight than plants with single PVM infection in most of the generations studied. The results indicate a strong negative impact of PVS in mixed infections with PVM and show the importance of its elimination from potato plants