Seedless barberry fruit is one of the important horticultural products of Iran, which has health benefits due to great amounts of phenolic compounds, flavonoids, and antioxidant activity. However, fresh barberry fruit has a short shelf life even at cold storage, mainly due to high water content and thin skin that leads to fungal decay and high postharvest loss. We examined the effectiveness of the postharvest ultrasonic technology on the quality preservation and nutritional value of fresh seedless barberry fruit and their decay reduction during cold storage. Experimental treatments were the time and temperature of ultrasound (US) and included: (1) control, fruit without US, (2) 5 min US at 20°C, (3) 5 min US at 30°C, (4) 5 min US at 40°C, (5) 10 min US at 20°C, (6) 10 min US at 30°C, (7) 10 min US at 40°C, (8) 15 min US at 20°C, (9) 15 min US at 30°C, and (10) 15 min US at 40°C. After applying the treatments, the fruits were sealed in polyethylene bags and stored at 4 ± 1°C for 20 days. The results showed that all US treatments had higher titratable acidity, antioxidant activity, phenol content, and vitamin C content than the control. However, the highest titratable acidity and antioxidant activity values were obtained in US treatments at 40°C and 30°C for 15 min. Also, US treatment significantly reduced the total soluble solids, decay percentage, and microbial load of fresh barberry fruit. As the US treatment temperature increased from 20°C to 40°C, the decay and microbial load of fruit significantly decreased. In conclusion, this study proved the potential application of the US for preserving the quality of fresh seedless barberry fruit, and the most optimal US temperature and its application time was 40°C for 15 min.