This work was carried out to study the effects of substitution fried oyster mushroom (Pleurotus ostreatus) by beef meat at levels (10, 20, 30 and 40%) and the effect of frying on chemical composition, physical, microbiological contamination and organoleptic properties of meatballs. The results showed that moisture, ash and carbohydrate contents of the raw meatballs were increased, but protein, fat contents and pH value were decreased by increasing levels of fried mushroom. As a result of frying, moisture was decreased, but protein, fat, ash and carbohydrate contents were increased compared to those of raw meatballs. The result showed that the physical properties as tenderness, plasticity and Feder values were increased, but, water holding capacity values were decreased by increasing levels of fried mushroom, on the other hand all properties were decreased as affected by frying compared to raw samples. In addition, the Feder value of raw or fried meatballs were less than 4, this means that these products had good quality. The total microbial count of raw meatballs was decreased by increasing levels of fried mushroom. Also, total microbial count of meatballs was decreased as affected by frying. Furthermore, both raw or fried meatballs were free of sporo-performing bacteria, Coliform group and Salmonella spp. Similarly, all the formulations of meatballs can be used for the preparation of acceptable organoleptic attributes. Also, the meatball containing 10% fried mushroom was the best sample compared to other samples.