High-intensity ultrasound could be an alternative to pasteurization for cheeses made with fresh raw milk, the properties of which must be preserved as part of their intangible cultural heritage, such as Panela cheese in Mexico. This research aimed to study the effect of the amplitude (50% and 100%) and application time (0, 5, and 10 min) of ultrasound treatment of fresh raw milk, on the yield and microbiological and physicochemical qualities of Panela cheese after 24 h of storage at 4 °C. The yield was increased to 24.29% with 10 min of ultrasonication, although the amount of exudate was higher in the ultrasonic product than in the control (20.33%). As the ultrasonication time increased, the yellowness (b*) increased significantly, while the hue angle decreased (with values close to 90°), resulting in evident yellow tones in cheeses made with milk treated for 10 min. The pH significantly increased from 6.6 to 6.74 with 5 min of ultrasound, but decreased to 6.37 with 10 min of ultrasonication. Although no significant differences were found in fat content, the protein significantly increased with 5 min of sonication, but it decreased markedly when ultrasound was applied for 10 min. Ultrasound treatment with amplitudes of 50% effectively decreased the counts of coliform bacteria regardless of ultrasonication time. However, the mesophilic bacteria increased by a 0.9 log with an amplitude of 100% and 10 min treatment. The results showed that ultrasound improved the yield and microbial, nutritional, and physicochemical properties of Panela cheese.