The gut-microbiota-brain axis influences neuroinflammation, neural development and behaviour such as sociability, memory and anxiety. To study these traits in vivo, especially during development or disease, it is crucial to analyse them over time and with multiple analyses in the same animal. With a growing understanding of the role of specific bacteria in neurodegenerative disease and behaviour, the demand for gnotobiotic mouse models has increased. However, maintaining stable hygienic conditions during behavioural testing is challenging, as exposure to conventional environments can alter the hygienic status of mice and affect behaviour. We established protocols to perform behavioural tests assessing memory, anxiety, exploration, learning and recognition under axenic conditions using flexible film isolators. Our study compared the behaviour of germ-free mice with mice carrying a defined minimal or moderately diverse microbiota. The results showed no effect of the microbiota on short- and long-term memory or novel object recognition. However, we showed that mice colonised with defined moderately diverse commensal bacteria exhibited more anxiety-like behaviour than germ-free mice. In addition, we showed that microbiota complexity is important, as only mice colonised with moderately diverse microbiota exhibited anxiety-like behaviour, allowing us to disentangle the contribution of specific microbial species or community interactions to this phenotype. This phenotype associated with differences in hippocampal and serum metabolic profiles between colonised and germ-free mice. We propose a novel approach to study rodent behaviour at different physiological and pathological stages in their life without compromising hygiene, thus promoting the refinement and reduction of mice used in experiments.