Little is known about how nicotinic signalling in human astrocytes may contribute to the functional neurotoxicity of compounds related to tobacco alkaloids and neonicotinoid pesticides. We generated a single-cell Ca²⁺-imaging assay in induced pluripotent stem cell (iPSC)-derived astrocytes, and profiled functional expressions of some neurotoxicologically relevant receptors. Responses to pharmacological tool compounds indicated the expression of nicotinic, muscarinic, purinergic, glutamatergic receptors and voltage-gated Na⁺/Ca²⁺ channels. Closer investigation of the nicotinic system, e.g., using the alpha7 nicotinic acetylcholine receptor (nAChR)-selective positive allosteric modulator PNU-120596 and alpha7-preferring agonist (AR-R17779) demonstrated that Ca²⁺ signals elicited by nicotine and neonicotinoids are dominated by alpha7 nAChRs and depend on the downstream activation of L-type Ca²⁺ channels and tetrodotoxin-sensitive Na⁺ channels. Crosstalk of nAChR activation/desensitization was not observed for the inflammatory response elicited by TNF or for activation of glutamatergic or purinergic signalling. However, pre-stimulation of nAChR by neonicotinoids significantly blunted the response to the neurotransmitter acetylcholine. Comparative experiments in the human neuronal cultures (LUHMES cells) revealed similar potency ranges and pharmacological fingerprints for several neonicotinoids and their human-relevant metabolites descyanothiacloprid and desnitroimidacloprid. The pesticide metabolites showed a high potency, compared with their respective parent compounds. After this basic system characterization, the hitherto data-poor pesticides cycloxaprid and flupyradifurone were comparatively profiled in astrocytic and neuronal test systems. They showed the typical features of alpha7 nAChR agonists. The disruption of cholinergic signalling in astrocytes suggests that neonicotinoids affect not only neurons in human brains. Therefore, future neurotoxicity screening approaches may need to consider astrocyte toxicity.