Fusarium culmorum is able to cause devastating crown rot disease, particularly in barley and wheat worldwide. The aim of this study was to investigate the early physiological and molecular changes in barley roots in response to F. culmorum infection. Therefore, we have infected 3-day old barley roots with a highly pathogenic F. culmorum isolate (F16). The root length and shoot length were significantly reduced at 7 days after infection in six widely cultivated Turkish barley cultivars. Based on the disease index values, Martı (six-rowed) and Tokak 157/37 (two-rowed) were selected. Defense response was comparatively assessed with measures including H2O2 production and induction of stress-induced genes at six-time points after infection (0–96 h). Fungal infection did not affect the membrane integrity of root cells while osmolality decreased and H2O2 production increased. At the molecular level, antioxidant-related genes, HvCu/ZnSOD, HvGST6, HvAPX and HvBAS1 were constitutively and strongly expressed unlikely to HvCAT2 in which transcript accumulation was slightly detected upon infection. Differential expression of HvMT2, HvLOX1 and HvWRKY12 has been observed following the infection. Importantly, pathogenesis related (PR) genes HvPR1, HvPR3, HvPR4, HvPR5 and HvPR10 were induced at different time points of infection. The transcript accumulation of HvPR4 was the highest while HvPR10 expressed in minimal levels. Our results showed unexpected cellular responses such as disruption of osmotic adjustment in barley roots and the role of PR genes in initial response under F. culmorum attack.