Two novel compounds were isolated for the first time from Calycotome spinosa (L.) Link, an alkaloid 5-Hydroxy-1H-indole (4) and a cyclitol D-pinitol (5), together with the three well-known flavonoids; Chrysin-7-O-(β-D-glucopyranoside) (1), Chrysin-7-O-β-D-(6″-acetyl)glycopyranoside (2) and Apigenin-7-O-β-D-glycopyranoside (3). The chemical structures of the isolated compounds were elucidated by spectroscopic data and mass spectrometric analyses; including a fresh approach 1D-NMR, 2D-NMR with LC-ESI-MS/MS. In this study, the new compound (4) that has been obtained from the leaves MeOH extract presented the best radical scavenging activity (DPPH) (IC50 < 10 µg/mL) compared to the standard butylated hydroxytoluene (BHT, IC50 = 34.73 ± 0.23 μg/mL) and showed the highest total antioxidant capacity (TAC = 985.54 ± 0.13 mg AAE/g extract) in contrast to ascorbic acid (TAC = 905.95 ± 0.07 mg AAE/g extract). Furthermore, the strongest reducing power (EC50 = 344.82 ± 0.02 µg/mL), as well as the remarkable scavenging potential by ABTS assay (IC50 = 7.8 ± 0.43 µg/mL), were exhibited by the same composite (4). Followed by the methanol crude extract and the compound (3) that also showed a potent antioxidant (DPPH; IC50 = 41.04 ± 0.15 and 47.36 ± 0.21 µg/mL, TAC; 671.02 ± 0.21 and 608.67 ± 0.34 mg AAE/g extract, FRAP; EC50 = 763.73 ± 0.32 and 814.61 ± 0.31 µg/mL, ABTS; IC50 = 19.18 ± 0.06 and 63.72 ± 0.64 µg/mL, respectively), but less than the previous samples. On the opposite side, compound (5) had the lowest activity, in which its values were less interesting to determine. Moreover, compound (4) has equally exerted an attractive antibacterial activity against Staphylococcus aureus (ATTC-25923), Pseudomonas aeruginosa (ATTC- 27853) and Salmonella abony (NCTC 6017), as measured by the disc diffusion assay, with inhibition zones of 16 ± 0.5, 9.83 ± 0.29 and 8 ± 0.28 mm, in that order. To the best of our knowledge, 5-Hydroxy-1H-indole was isolated from plants for the second time in our current work. Thus, the obtained results from this investigation propose that the leaves of C. spinosa are a rich natural source for value molecules as potential antioxidants and antimicrobial agents for best human health.