Bacopa monnieri and Bacopa floribunda are extensively utilized in various Ayurvedic remedies because of their cognitive-enhancing properties. Phytochemicals, antioxidants, anti-diabetic, anti-acetylcholine esterase activities, and the detection of bioactive substances (triterpenoid saponins and stigmasterol) were examined using HPLC and HR-LC-MS. The results showed that the aqueous extract of B. floribunda had the highest TPC (60.44 ± 5.24 mg TAE/g extract), while the acetonitrile extract had the lowest content (9.35 ± 0.64 mg TAE/g extract). The highest TFC was found in the acetone extracts of B. monnieri and B. floribunda (63.69 ± 13.48 and 61.44 ± 2.94 mg CE/g extract, respectively). Additionally, the acetone extracts of B. floribunda and B. monnieri revealed the greatest TTC (256.8 ± 7.73 mg CE/g extract) and TSC (363.5 ± 10.6 mg DE/g extract), respectively. In antioxidant studies, the acetone and methanol extracts of B. monnieri showed the highest DPPH radical scavenging, FRAP, and MC activities (68.24 ± 1.31 mg AAE/g extract, 160.43 ± 4.34 mg Fe(II)/g extract, and 283.13 ± 1.46 mg EDTA/g extract, respectively). The acetone extract of B. floribunda had better ABTS radical scavenging and PMA (13.29 ± 0.53 mg TE/g extract and 127.7 ± 0.51 mg AAE/g extract, respectively). The acetonitrile extract of B. floribunda showed considerable inhibitory effects against α-amylase (71.89 ± 10.1%), α-glucosidase (93.89 ± 0.19%), and acetylcholine esterase (80.92 ± 0.6%) enzymes. The presence of stigmasterol and four triterpenoid saponins were also confirmed by HPLC. The total amount of triterpenoid saponins was found higher in B. floribunda (4.97 ± 0.012 mg/g DW) than B. monnieri (3.96 ± 0.012 mg/g DW). In contrast, B. monnieri (5.13 ± 0.12 mg/g DW) exhibited the highest stigmasterol content as compared to B. floribunda (3.97 ± 0.35 mg/g DW). For the first time, we report B. floribunda as a potent source of triterpenoid saponins and could be a substitute for B. monnieri.