A new series of 12 examples of spiro[chromeno[4,3-b]thieno[3,2-e]pyridine]-7-amines tacrine hybrids (3) were designed and synthesized as cholinesterase inhibitors. These new compounds were synthesized at 20-33% yields by a one-pot two-step cyclocondensation reaction of 2-aminothiophene-3-carbonitriles (1) with spiro[chroman-2,10-cycloalkan]-4-ones (2) using AlCl3 as the catalyst without solvent and under conventional thermal heating. Subsequently, these new tacrine hybrids were subjected to AChE and BChE inhibitory activity evaluation and molecular docking studies. In vitro cholinesterase assays and in silico docking indicated that all new tacrine analogs 3 were not AChE inhibitors. However, the molecules without a carbocyclic moiety geminated to the thiophene ring, i.e., spiro scaffolds that originated from cyclopentanone (3aa), cyclohexanone (3ba), and cycloheptanone (3ca) showed the highest inhibitory potency against the BChE enzyme, thereby proving to be promising candidates for evaluation in Alzheimer's disease synthetic models.