This study aimed to determine the effect of ultrasound (in two different frequencies of 30, 42 kHz) on the physical, biochemical, anti-oxidant, and antimicrobial properties of industrial Iranian honey. All samples (except control) were treated using 30 or 42 kHz ultrasound at (20 and 45°C for 1, 5, and 10 minutes). Then the changes related to 5-hydroxymethylfurfural (HMF) content, pH, acidity, proline, total count of aerobic mesophilic bacteria, diastase, moisture, sucrose, fructose, glucose, the ratio of fructose to glucose, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), osmophile, phenol, regenerating sugars and total sugars were evaluated on days 1, 30, 90, and 180. In both treatment groups (affected by 30 or 42 kHz ultrasound) there were physical, biochemical, antioxidant, and antimicrobial parameter changes. These changes in the group treated with 42 kHz ultrasound were reduced with a decrease in humidity, pH, acidity, glucose-fructose ratio, sugar, clostridium, total microbial count, mold, osmophiles, HMF, and an increase in diastase, proline, and phenol. In both treatment groups, the changes were more significant on days 90 and 180. Using 42 kHz ultrasound at 45 ºC for 10 minutes could lead to an increase in the quality and shelf life of industrial honey. Therefore, this technique can be used in the food industry to provide a higher quality product as well as a more successful export.