In India, stingless bee propolis has not yet been received commercial importance due to lack of information about its chemical composition and health benefits. Therefore, herein, we comprehensively studied the chemical composition and biological activities of seven Indian stingless bee propolis samples collected from four different states. Ethanolic extracts of stingless bee propolis (EESP) samples were prepared and their yield, total phenolic and flavonoid content were estimated. Antioxidant activities of EESP samples were assessed using DPPH, ABTS, and FRAP assays. Similarly, antibacterial (against both gram-positive and gram-negative bacteria) and antifungal activities of all EESP were studied using a 96-well plate microdilution method. HPLC and 1HNMR techniques were used for quantitative and fingerprint profile analysis. Results showed that the studied EESP samples had considerable antioxidant and antimicrobial activities. The observed antioxidant and antimicrobial activities had positive and negative correlations with total phenolic and flavonoid content of EESP, respectively. HPLC and 1HNMR studies showed that Indian stingless bee propolis is of a “gallic acid-naringin” type of unique propolis, which may also contain other compounds such as sugars, terpenes, and steroids. The chemometric analysis of 1HNMR further demonstrated that the chemical composition of Indian stingless bee propolis samples varies with collection site, and samples from the same or adjacent region were chemically more similar. In conclusion, the present study is the first report on biological and chemical standardization of Indian stingless bee propolis, which will help to commercialize stingless bee propolis in the healthcare market.