Background: Helicteres isora is a highly valued medicinal plant whose fruits have been traditionally used throughout Asia, Africa, and other parts of the world in treating a variety of diseases, including diabetes mellitus and memory-related health problems. This study aimed to evaluate the in-vitro/in-vivo toxicity and anti-amnesiac profile of Helicteres isora (H. isora) extract. Methods: Initially, experimental animals were subjected to acute, sub-chronic, and chronic toxicity evaluation to establish the safety profile of the H. isora-crude (Hi-Crd) extract of selected plant fruit extract. In the first phase, oral administrations of the graded doses of 500, 1000, and 1500 mg/kg body weight (b.w.) were administered to experimental rats for 14 days. In the second phase, higher doses (3000, 4000, and 5000 mg/kg b.w.) were administered to assess acute toxicity. On the 28th day, blood was obtained and subjected to serum biochemical analysis and lipid profiling to evaluate chronic toxicity. To further evaluate the toxicity in deep, histopathology studies of the kidney, liver, and pancreas were performed. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities of the extract were also performed, and IC₅₀ values were calculated. The scopolamine-induced amnesic mice were utilized in behavioral studies (Y-maze and Novel Object Recognition Test (NORT)), where doses of 75 and 150 mg/kg b.w. of Hi-Crd were administered to experimental animals. Results: In the acute toxicity test, no symptoms of morbidity or toxicity were observed. Hi-Crd increased the relative liver and kidney weight at the end of the sub-chronic toxicity test when given at a dose of 4000 mg/kg b.w.. During the chronic toxicity test, the extract showed substantial effects on several biochemical parameters. Although a rise in the lipid profile was noted, the overall effect on other biochemical parameters was negligible. At day 28, there was no significant effect on the liver enzyme markers, and thus the extract was considered to be safe at tested doses. Hi-Crd has a safe profile in terms of uric acid, bilirubin, and blood urea while there was a slight rise in the serum creatinine level. The H.isora-chloroform fraction (Hi-Chl) showed maximum activity with IC₅₀ values of 54.29 ± 1.17 μg/mL against AChE and 105.66 ± 2.91 μg/mL against BuChE. In behavioral studies, the chloroform extract considerably enhanced spontaneous alteration performance as measured through the Y-maze test (p < 0.001), at tested doses. Its counterpart, the ethyl acetate fraction, also showed noteworthy outcomes with good significance levels; p < 0.01 and p < 0.001 at 75 and 150 mg/kg b.w., respectively. In addition, the chloroform fraction significantly improved the discrimination index (DI) in experimental mice to 67.08% as compared to the amnesic group (29.87%) mice results. Conclusions: Helicteres isora Linn was found to be well tolerated during the performed set of tests and could be considered an effective treatment to enhance memory impairment in short- and long-term therapy with an adequate margin of safety. However, the study found that it may have adverse effects to an imperceptible level on kidney and liver functions. Therefore, further studies on other animal models are necessary to confirm its future use as a drug.