Siraitia grosvenorii fruit (SGF) has been used as a natural sweetener and traditional medicine in China for more than two centuries. This study evaluated the effect of SGF extract supplementation (0.5%, 1%, and 2%) on the chemical, microbial and sensory properties of probiotic yogurt. The antioxidant, angiotensin-converting-enzyme inhibitory (ACE-I) and antibacterial bioactivities were determined. SGF extract supplementation improved some of the chemical and physicochemical characteristics. Probiotic yogurt with the fruit extract had significantly more Lactobacillus casei and Lactobacillus bulgaricus, whereas there was no significant effect on the number of Streptococcus thermophiles. The bioactivities were significantly increased by SGF extract supplementation. Probiotic yogurt with 2% SGF extract showed the highest antioxidant, ACE-I, and antibacterial activities, whereas the one with 1% SGF extract conferred the highest sensory attributes score. Overall, SGF extract offers a promising option as a dietary supplement to produce novel dairy products that have high nutritional and bioactivity values.