The experiment was conducted on a farm in the AL-Bandar region, southwest of Al-Muthanna governorate (3 km from Samawah center), during the 2022-2023 agricultural season. To determine the effect of four levels of Azolla extract (0, 15, 30, 45 liters ha-1 ) on three barley Cultivars (Axad, IPA 99, IPA 265). The study used a split-plot design with three replications using a complete randomized block design (C.R.B.D) where Azolla inhabited the secondary plots while the Cultivar occupied the primary ones. The results of the experiment showed that the 30 liters ha-1 level was significantly superior in spikes number per m 2 , 1000 grain weight, and grain yield, giving averages of (484.60 spikes m 2 ), (45.91 g), and (6.01 t ha-1 ) respectively. The 45 liters ha-1 level was superior in grain protein content, giving an average of (12.37%). Also, the Axad Cultivar was greater in spikes number of m2 , grains number in the spike, grain yield, and grain protein content, giving averages of (492.50 spikes m2 ), (47.97 grains spike - 1 ),(6.05 t ha-1 ), and (12.06%) respectively. The IPA 265 Cultivar was superior in 1001 grain weight, giving an average of (46.02 g). The results showed that the 45 liters ha-1 level of Azolla extract with the Axad Cultivar for the combination (V1 × A3) was significantly superior in the spikes number and grain protein content, giving averages of (528.20 spikes m2 ) and (12.86%) respectively.