Arecoline is a naturally occurring psychoactive alkaloid with partial agonism at nicotinic and muscarinic acetylcholine receptors. Arecoline consumption is widespread, making it the fourth (after alcohol, nicotine and caffeine) most used substance by humans. However, the mechanisms of acute and chronic action of arecoline in-vivo remain poorly understood. Animal models are a valuable tool for CNS disease modeling and drug screening. Complementing rodent studies, the zebrafish (Danio rerio) emerges as a promising novel model organism for neuroscience research. Here, we assessed the effects of acute and chronic arecoline on adult zebrafish behavior and physiology. Overall, acute and chronic arecoline treatments produce overt anxiolytic-like behavior without affecting general locomotor activity and whole-body cortisol levels. Acute arecoline at 10 mg/L disrupted shoaling, increased social preference, elevated brain norepinephrine and serotonin levels and reduced serotonin turnover. Acute arecoline also upregulated early protooncogenes c-fos and c-jun, whereas its chronic treatment elevated brain expression of microglia-specific biomarker genes egr2 and ym1 (thus, implicating microglial mechanisms in potential effects of long-term arecoline use). These findings support high sensitivity of zebrafish screens to arecoline and related compounds, and reinforce the growing utility of zebrafish for probing molecular mechanisms of CNS drugs. Our study suggests that novel anxiolytic drugs can eventually be developed based on arecoline-like molecules, whose integrative mechanisms of CNS action may involve monoaminergic and neuro-immune modulation.