Alzheimer’s disease (AD) is the most common and severe form of dementia. Worldwide, it is estimated that 46.8 million people suffer from dementia and by 2050, this number is expected to reach approximately 131.5 million people due to increasing numbers of elderly people. One of the neuropathological features of AD is represented by the degeneration of cholinergic neurons in the basal forebrain. At the cognitive level, the main hallmark of AD is memory decline. Nicotinic acetylcholine receptors (nAChRs) modulate the neurobiological processes underlying hippocampal learning and memory. Nicotine stimulates nAChRs thus improving the attention, memory and learning. However, nicotine’s cardiovascular and addictive side-effects have limited its therapeutic use in AD but remain a strong scaffold for developing new drugs for AD. Cotinine (COT) and 6-hydroxy-L-nicotine (6HLN), two nicotine derivatives that are structurally similar, were found to possess antioxidant and cognitive-enhancing properties without showing the side-effects of their precursor. In this study, we used in silico tools to evaluate and compare the binding potential of COT and 6HLN in two different allosteric binding sites (α4-α4 and α4-β2) of human α4β2 nAChRs (PDB ID 6CNK). We have also performed a series of in vivo tasks to assess the effects of COT and 6HLN on memory impairment in a rat model of AD induced by brain infusion of Aβ25-35 peptide. The acetylcholinesterase (AChE) activity was also measured. Our results showed that COT and 6HLN bind preferentially and with higher energy than nicotine to α4-β2 compared to α4-α4 interface of α4β2 nAChRs. COT and 6HLN administration mitigate the spatial and recognition memory deficits and decreased the specific activity of AChE in the hippocampus of Aβ25-35-treated rats. These results suggest that COT and 6HLN might represent new therapeutic agents in AD by modulating cholinergic activity. This work was supported by a grant of CNCS-UEFISCDI, project number PN-III-P1-1.1-TE2016-0367, within PNCDI III.