Background Invasive plant species pose a significant threat for fragile isolated ecosystems, occupying space, and consuming scarce local resources. Recently though, an additional adverse effect was recognized in the form of its secondary metabolites entering the food chain. The present study is elaborating on this subject with a specific focus on the Nicotiana glauca Graham (Solanaceae) alkaloids and their occurrence and food chain penetrability in Mediterranean ecosystems. For this purpose, a targeted liquid chromatography electrospray tandem mass spectrometric (LC-ESI-MS/MS) analytical method, encompassing six alkaloids and one coumarin derivative, utilizing hydrophilic interaction chromatography (HILIC) was developed and validated. Results The method exhibited satisfactory recoveries, for all analytes, ranging from 75 to 93%, and acceptable repeatability and reproducibility. Four compounds (anabasine, anatabine, nornicotine, and scopoletin) were identified and quantified in 3 N. glauca flowers extracts, establishing them as potential sources of alien bio-molecules. The most abundant constituent was anabasine, determined at 3900 mu g/g in the methanolic extract. These extracts were utilized as feeding treatments on Apis mellifera honeybees, resulting in mild toxicity documented by 16-18% mortality. A slightly increased effect was elicited by the methanolic extract containing anabasine at 20 mu g/mL, where mortality approached 25%. Dead bees were screened for residues of the N. glauca flower extracts compounds and a significant mean concentration of anabasine was evidenced in both 10 and 20 mu g/mL treatments, ranging from 51 to 92 ng/g per bee body weight. Scopoletin was also detected in trace amounts. Conclusions The mild toxicity of the extracts in conjunction with the alkaloid and coumarin residual detection in bees, suggest that these alien bio-molecules are transferred within the food chain, suggesting a chemical invasion phenomenon, never reported before.