The aim of this work was to evaluate the effect of replacing wheat flour with wheat germ (10 and 20%) or fat by different fat replacers, including inulin (20 and 40%) and chia mucilage (10, 15, and 20%) on the quality attributes and physicochemical properties of biscuits. The results showed that the wheat germ substitution significantly (P≤ 0.05) increased the protein, ash, and crude fiber content of biscuits compared to the control sample. The fat content progressively increased with the increment substitution levels of wheat germ. Moreover, the fat content gradually decreased with the increase in the fat replacers content. There were no significant differences (P≤ 0.05) in the protein content between the control sample and other samples prepared with different ratios of fat replacers. The results also indicated that the fat replacement by inulin or chia mucilage reduced the specific volume of the biscuit compared to the control sample. Our findings indicated that utilizing inulin by 30 and 40% significantly reduced fat content by 45.32 and 58.33%, however 20% chia mucilage decreased fat content by 22.36% in biscuit samples. Reducing the fat content by inulin and chia mucilage as fat replacers increased the hardness and decreased the fracturability values of the produced biscuits compared to the control sample. According to the organoleptic findings, there were no significant differences (P≤ 0.05) between the control and other treated samples in terms of color and texture criteria. Using chia mucilage as a fat replacer at a level of 20% improved the overall acceptability of the biscuit compared to the control sample.