Ultrasound is one of the most promising non-thermal an emerging technique in food technology. The objective of the present work was to evaluate the effect of different ultrasonic treatments on the most important wine microbiota (Saccharomyces and non-Saccharomyces yeasts and lactic acid bacteria). Two stages were carried out: the assessment step, where six different ultrasonic treatments (with varying power, time, and pulses) were used on Saccharomyces cerevisiae, Brettanomyces spp., and Lactiplantibacillus plantarum; and the validation step, where two chosen ultrasonic treatments were used on Zigosaccharomyces bailli, Brettanomyces spp., Saccharomyces cerevisiae, Saccharomyces bayanus, Pichia membranifaciens, Schizosaccharomyces pombe, and Hanseniaspora osmophila. The most sensitive microorganism was Brettanomyces spp., and the most resistant was Lactiplantibacillus plantarum. Ultrasonic treatments had varying effects on vitality (delay of growth or maximum OD reduction) and on viability (reduction of microbial growth).