A pot experiment was conducted in two seasons 2020 and 2021 at wire green house of Plant Physiology Department, Cotton Research Institute, Agricultural Research Center, Giza, Egypt, to evaluate the effect of using azolla extract (AE) at concentration of 30% on cotton plants Giza 95 cultivar under different salinity water irrigation (SWI). The experiment design was split-plot, which the main plots were devoted to SWI treatments (0, 8000 and 12000 ppm), while AE at concentration of 30% applications were randomly occupied the subplots including control (C), azolla seed soaking (ASS), azolla foliar application (AFA) and azolla combination application (ACA) of ASS+AS treatments. The results noticed that AE treatments improved the plant growth characters, chemical constituents of cotton leaves (total chlorophyll, carotenoids, total soluble sugars, total phenols, total free amino acids, proline and total soluble proteins), in addition to increase the enzymes activity of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and glutathione reductase (GR), total antioxidant capacity and yield characters, while fiber properties did not affected when compared with untreated plants. Cotton plants treated with ACA gave the maximum values of seed cotton yield/plant about 39.09 and 27.45%, number of bolls/plant about 24.74 and 25.66% and boll weight about 11.40 and 10.52% in both seasons, respectively, compared to untreated plants. The plants treated with SWI (8000 and 12000ppm) had reduced pigments content, growth and yield than plants under normal condition (0ppm). AE treatments by ACA gave the best results in leaves chemical constituents, enzymes activity, growth, yield characters, and improving salinity tolerance in cotton plants under different SWI concentration.