This study investigated the effects of using defatted corn germ flour (DCGF) in preparing tortilla chips on the physicochemical, texture, microstructure, antioxidant, and sensory properties of the final product. Defatted corn germ flour was added to tortilla chip formulations at 0%, 5%, 10%, and 15%. The results showed that fortification with DCGF increased the protein and ash contents and decreased the carbohydrate content of tortilla chips compared to the control samples (100% corn flour), while the fat content was not significantly affected even with the addition of 10% DCGF. Additionally, fortification with DCGF increased tortilla chips' essential amino acid content, total phenolic, total flavonoids, and antioxidant activity. Fortification at 5% and 10% DCGF reduced the water-holding capacity of tortilla chips compared to the control sample, while their oil-holding capacity was not significantly affected. The hardness of tortilla chips decreased after fortification with 5% and 10% DCGF and increased with 15% DCGF. Higher cohesiveness, springiness, gumminess, chewiness, and fracturability values were observed in the 15% DCGF samples. According to the sensory evaluation of tortilla chips, fortification with 15% DCGF was found to be acceptable by participants, but the highest sensory acceptance rates were observed in the 5% and 10% DCGF samples. Therefore, this study recommends the use of DCGF as a nutritional and functional ingredient at 5% and 10% in the preparation of tortilla chips.