Numerous studies have reported that prolonged or multiple exposures to anaesthetics in early life lead to detrimental effects on brain function, most having focused on neurocognitive function, and relatively few on long term neuropsychiatric performance. The present study investigated the impact of repeated neonatal isoflurane exposure on chronic variable stress (CVS)-induced psychiatric and behavioural outcomes together with CVS-related neuronal activity and neuro-inflammatory reactivity in relevant brain circuits. In the present study, C57BL/6J mice received either three exposures to isoflurane at postnatal days 7, 8, and 9 or a control exposure. From postnatal day 45, mice were exposed to a mild, 3-week, CVS paradigm or none and the CVS-related neuropsychiatric performance was evaluated using a series of behavioural tests. The neuronal activity in relevant brain regions was measured by ΔFosB immunopositivity and CVS-related neuroinflammation was assessed by analysing levels of pro-inflammatory cytokines IL-1α, IL-1β, IL-6, and TNF-α. In mice experiencing serial neonatal isoflurane exposure, we detected a significant enhancement in anxiety levels following CVS procedures, together with enhanced neuronal activity, and exacerbated neuroinflammation in the basolateral amygdaloid nuclei (BLA) and hippocampal dentate gyrus (DG) regions. No such change was found in control mice. These results indicate an association between early multiple isoflurane exposures in infant mice and susceptibility to a CVS-evoked anxious phenotype accompanied by enhanced neuronal activity in BLA and DG regions and high inflammatory reactivity in response to CVS.