Introduction: The jellyfish Cassiopea has a conspicuous lifestyle, positioning itself upside-down on sediments in shallow waters thereby exposing its photosynthetic endosymbionts (Symbiodiniaceae) to light. Several studies have shown how the photosymbionts benefit the jellyfish host in terms of nutrition and O₂ availability, but little is known about the internal physico-chemical microenvironment of Cassiopea during light–dark periods. Methods: Here, we used fiber-optic sensors to investigate how light is modulated at the water-tissue interface of Cassiopea sp. and how light is scattered inside host tissue. We additionally used electrochemical and fiber-optic microsensors to investigate the dynamics of O₂ and pH in response to changes in the light availability in intact living specimens of Cassiopea sp. Results and discussion: Mapping of photon scalar irradiance revealed a distinct spatial heterogeneity over different anatomical structures of the host, where oral arms and the manubrium had overall higher light availability, while shaded parts underneath the oral arms and the bell had less light available. White host pigmentation, especially in the bell tissue, showed higher light availability relative to similar bell tissue without white pigmentation. Microprofiles of scalar irradiance into white pigmented bell tissue showed intense light scattering and enhanced light penetration, while light was rapidly attenuated over the upper 0.5 mm in tissue with symbionts only. Depth profiles of O₂ concentration into bell tissue of live jellyfish showed increasing concentration with depth into the mesoglea, with no apparent saturation point during light periods. O₂ was slowly depleted in the mesoglea in darkness, and O₂ concentration remained higher than ambient water in large (> 6 cm diameter) individuals, even after 50 min in darkness. Light–dark shifts in large medusae showed that the mesoglea slowly turns from a net sink during photoperiods into a net source of O₂ during darkness. In contrast, small medusae showed a more dramatic change in O₂ concentration, with rapid O₂ buildup/consumption in response to light–dark shifts; in a manner similar to corals. These effects on O₂ production/consumption were also reflected in moderate pH fluctuations within the mesoglea. The mesoglea thus buffers O₂ and pH dynamics during dark-periods.