Boron-containing compounds (BCCs) have emerged as a new class of organic molecules in pharmaceutical chemistry as novel drug candidates for extensive therapeutic applications. In this context, the five novel bioactive spiroborate compounds (S1-S5) were synthesized by condensation reaction of Schiff base ligands (L1-L5), 1,3-dioxane-5,5-dimethanol, and boric acid with high yields. The bioactive spiroborate compounds (S1-S5) were characterized by NMR (1H and 11B) spectra, FT-IR spectra, UV–Vis spectra, LC-MS/MS spectrometry, elemental analysis, and melting point measurement techniques. The anti-cancer activity in A549 lung cancer cells and inhibitory effects of α-glucosidase with acetylcholinesterase (AChE) enzymes of the synthesized spiroborate compounds were investigated. In addition, the anti-bacterial activity of spiroborate compounds against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli were investigated, respectively. On the other hand, anti-biofilm activity against Pseudomonas aeruginosa was assessed. In silico studies were also conducted on the synthesized spiroborate molecules (S1-S5). Among the synthesized spiroborate compounds, spiroborate (B5) was identified as the most potent anticancer agent with an IC50 of 352.5 µg/ml, in contrast spiroborate (B3) exhibited the strongest α-glucosidase inhibition (68 %) and anti-bacterial activity. Furthermore, spiroborate (B4) was the most effective AChE inhibitor (61 %). Besides, spiroborate (B4) exhibited strong antibacterial activity against S. aureus and E. coli with MIC values of 7.81 μg/mL. Spiroborate compounds (S1-S5) exhibited significant dose-dependent biofilm inhibitory activity against P. aeruginosa. The binding affinities of spiroborate (B4) to the proteins 3VSL and 4WUB were calculated as −4.16 and −4.26 kcal/mol.