In order to develop effective anti‐cholinesterase compounds, a novel series of pyrano[3’,4’:5,6]pyrano[2,3‐b]quinolinones were designed, synthesized, and evaluated in vitro against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). All derivatives showed very good AChE inhibitory (AChEI) activity (IC₅₀ = 0.37–5.62 μM) compared with rivastigmine (IC₅₀ = 11.07 μM). Among them, 11‐amino‐12‐(2,3‐dichlorophenyl)‐3‐methyl‐7,8,9,10‐tetrahydropyrano[3’,4’:5,6]pyrano[2,3‐b]quinolin‐1(12H)‐one (6f) displayed the best inhibitory activity. However, most of the synthesized compounds showed no anti‐BChE activity and compounds 6b and 6f were found to be only moderate inhibitors. The most potent anti‐AChE compound 6f had low and moderate inhibitory activity and neuroprotective effects against beta‐secretase (BACE1) and oxidative stress‐induced cell death, respectively. Also, kinetic and molecular docking studies of binding interactions elucidated that compound 6f bound to both the catalytic anionic site (CAS) and peripheral anionic site (PAS) of AChE.