The endocannabinoid system (ECS) has gained attention as a potential target to treat Alzheimer’s disease (AD) due to its neuroprotective, antioxidant, and anti-inflammatory properties. Consequently, natural cannabinoids like delta9-tetrahydrocannabinol (THC) and cannabidiol (CBD) have emerged as potential drugs against AD. Previous findings from our research group demonstrated that a combination of non-psychoactive doses of THC and CBD mitigates cognitive decline in an AD murine model, the APP/PS1 mice. Cognitive decline in AD is linked to the dysregulation of glutamate transmission within the hippocampus. In this sense, we observed by in vivo microdialysis techniques that chronic THC+CBD treatment reduces excessive hippocampal glutamate levels in APP/PS1 animals. However, in vivo microdialysis has limited temporal resolution, constraining in-depth study of cannabinoid effects at the molecular level. To overcome these limitations, in the present study we set up fiber photometry techniques to evaluate the impact of THC and CBD on glutamate dynamics and neuronal activity in the hippocampus of APP/PS1 mice with a millisecond resolution. Using fluorescent biosensors, we evaluated acute effects of THC, CBD, or THC+CBD on glutamate release and neuronal activity (i.e. cytosolic calcium levels) in response to hippocampal electrical stimulation in anesthetized WT and APP/PS1 animals. We found that THC induces a higher glutamate release only in APP/PS1 animals when stimulating at high frequencies, while its combination with CBD reverts this effect. Besides, neuronal activation is reduced when both cannabinoids are present, revealing a synergistic effect of THC and CBD that could be of interest within this context of hyperexcitability.