Abstract: This study aimed to evaluate the potential use of Agaricus bisporus mushroom as a natural antioxidant, fat/salt substitute and flavor enhancer in beef burger. Ten treatments were considered using a fractional factorial design (salt [0.5, 1.25 and 2.0%], fat [10, 15 and 20%] and mushroom content [0, 15 and 30%]). Treatments were characterized by instrumental and sensory measurements. The statistical design indicated that fat content had little influence on the results. The principal component analysis showed that the incorporation of mushrooms and salt modified the texture, moisture and water activity. Oxidative stability decreased as the salt content increased, at a low-fat content. Finally, the sensory profile was affected by the mushroom and salt contents, with the fat content exerting the least influence. Therefore, mushroom incorporation in beef burger may be a feasible strategy to reduce the fat content of beef burgers. Graphical abstract: [Figure not available: see fulltext.]