A series of bezofuran appended 1,5-benzothiazepine compounds 7a-v was designed, synthesized and evaluated as cholinesterase inhibitors. The biological assay experiments showed that most of the compounds displayed a clearly selective inhibition for butyrylcholinesterase (BChE), while a weak or no effect towards acetylcholinesterase (AChE) was detected. All analogs exhibited varied BChE inhibitory activity with IC50 value ranging between 1.0 ± 0.01 and 72 ± 2.8 μM when compared with the standard donepezil (IC50, 2.63 ± 0.28 μM). Among the synthesized derivatives, compounds 7l, 7m and 7k exhibited the highest BChE inhibition with IC50 values of 1.0, 1.0 and 1.8 μM, respectively. The results from a Lineweaver-Burk plot indicated a mixed-type inhibition for compound 7l with BChE. In addition, docking studies confirmed the results obtained through in vitro experiments and showed that most potent compounds bind to both the catalytic anionic site (CAS) and peripheral anionic site (PAS) of BChE active site. The synthesized compounds were also evaluated for their in vitro antibacterial and antifungal activities. The results indicated that the compounds possessed a broad spectrum of activity against the tested microorganisms and showed high activity against both gram positive and gram negative bacteria and fungi.