Abstract. Susmiati, Melia S, Purwati E, Alzahra H. 2022. Physicochemical and microbiological fermented buffalo milk produced by probiotic Lactiplantibacillus pentosus HBUAS53657 and sweet orange juice (Citrus nobilis). Biodiversitas 23: 4329-4335. Fermented milk is a type of dairy product that has been fermented with lactic acid bacteria. Changes in starter lactic acid bacteria and fruits can improve fermented dairy's physicochemical quality and total lactic acid bacteria (LAB) colonies. This study investigates how changes in the concentration of starter Lactiplantibacillus pentosus HBUAS53657 and sweet orange juice concentration affect protein, fat, moisture, total titratable acidity, pH value, and total lactic acid bacteria (LAB) colonies in buffalo fermented milk. This study used randomized block design (RBD) experiment, with factor A being starter L. pentosus strain HBUAS53657 4%, 5% and 6% (A1, A2 and A3) and factor B being sweet orange juice 10%, 15% and 20% (B1, B2 and B3) with three replications as a treatment. The data were statistically processed using analysis of variance, and the smallest significant difference test was p<0.05. The results showed that the protein content ranged from 5.81-6.33%, fat content 6.14-6.35%, water content 81.63- 85.78%, pH 3.57-4.23, TTA value 1.30-1.85 and total LAB colonies from 4.67×109 to 9.0×109 CFU/mL. Fermented milk products with 6% starter L. pentosus HBUAS53657 and 20% sweet orange juice produced the best results. This product is suitable for consumption as a probiotic beverage.