Aims Evaluate the effect of varying the droplet size of microspheres charged with thyme essential oil (TEO-MS) on their swelling (Sw), release rate (%RR) and in vitro antifungal activity against Saprolegnia sp. Methods TEO-MS obtained by ionic gelation were characterised through SEM microscopy and X-ray microtomography. Their Sw and RR% were evaluated at simulated fish-gastrointestinal conditions using gravimetric and spectrophotometric techniques. Results For all evaluated droplet sizes (p >= 0.05), TEO was heterogeneously distributed inside of the MS and TEO-MS experimented agglomeration and sphericity loss after the drying process. Under gastric conditions, the acid pH (2.9) limited the Sw (50-100%) of TEO-MS, generating a low RR% (14-18%). Contrary, the slightly alkaline intestinal pH (8.1) favoured the Sw (similar to 3.2 to 3.8 times) and therefore the RR% (42-63%). Conclusions TEO-MS (5-100 mg/mL) presented antifungal capacity onto Saprolegnia sp. after the simulated fish digestion, being the small droplet size once the most effective.