Scientific research on the impact of microplastics (MPs) in terrestrial systems is still emerging, but it has confirmed adverse health effects in organisms exposed to plastics. Although recent studies have shown the toxicological effects of individual MPs polymers on honey bees, the effects of different polymer combinations on cognitive and behavioural performance remain unknown. To fill this knowledge gap, we investigated the effects of oral exposure to spherical MPs on cognitive performance and brain accumulation in the honey bee Apis mellifera. We evaluated the acute toxicity, after a two-day exposure, of polystyrene (PS - 4.8-5.8 μm) and plexiglass (Poly(methyl methacrylate), or PMMA - 1-40 μm) MPs, and a combination of the two (MIX), at two environmentally relevant and one higher concentration (0.5, 5 and 50 mg L−1) and analysed their effects on sucrose responsiveness and appetitive olfactory learning and memory. We also used fluorescent thermoset amino formaldehyde MPs (1–5 μm) to explore whether microspheres of this diameter could penetrate the insect blood-brain barrier (BBB), using Two-Photon Fluorescence Microscopy (TPFM) in combination with an optimized version of the DISCO clearing technique. The results showed that PS reduced sucrose responsiveness, while PMMA had no significant effect; however, the combination had a marked negative effect on sucrose responsiveness. PMMA, PS, and MIX impaired bee learning and memory in bees, with PS showing the most severe effects. 3D brain imaging analysis using TFPM showed that 1–5 μm MPs penetrated and accumulated in the brain after only three days of oral exposure. These results raise concerns about the potential mechanical, cellular, and biochemical damage that MPs may cause to the central nervous system.