The population of Croatian autochthonous cultivars has a high degree of infection with economically important viruses, so it is necessary to carry out the elimination of the viruses in some cultivars to obtain healthy planting material. In this research, we tested in vitro meristem culture establishment on 18 autochthonous cultivars with different viral infections and the possibility of GLRaV-3 elimination through in vitro meristem culture. Plant material was sampled in a vineyard in two phenological stages, 10 days before flowering and 10 days after flowering of the grapevine. Apical meristem explants (1 mm) were placed into the MS culture medium supplemented with 0.5 mg/L benzyl adenine (BA) and 0.05 mg/L indol-3-acetic acid (IAA), and their survival, regeneration, and rooting were monitored. The results showed that the cultivar and the growth phase have a significant impact on the success of in vitro culture. In all cultivars studied higher success of in vitro culture establishment parameters (survival, regeneration, and rooting) was obtained in the case of explants sampled after flowering, with the exception of one cultivar for explants survival. Contrary to expectations, genotypes infected with three viruses (GLRaV-1, GLRaV-3, and GFLV) showed better results than genotypes infected with one or two viruses. The results showed successful in vitro establishment of Croatian autochthonous cultivar and GRLaV-3 elimination in one cultivar. However, due to the significant effect of cultivar, for routine application of this in vitro protocol on more than 100 autochthonous cultivars in need of sanitation, further studies should be conducted.