As fish are poikilothermic animals, i.e. the temperature of their body is dependent on the external environment, we aimed to investigate the effect of temperature on the in vitro adhesive ability of potential fish probiotics. The tested strains were Bifidobacterium animalis Bb12, Lactobacillus rhamnosus GG, L. rhamnosus LC 705, L. rhamnosus LCR 1/83 and Enterococcus faecium M74. The in vitro adhesive ability of the five strains to two types of rainbow trout mucus (skin and intestinal mucus) was determined at temperatures ranging from 4°C to 25°C. Three of the tested strains, B. animalis Bb12, L. rhamnosus GG and L. rhamnosus LCR 1/83, showed high adhesion to both types of mucus at all temperatures studied (19–30% and 11–29% of the added bacteria to skin and intestinal mucus, respectively). The adhesive abilities of the remaining strains were low in comparison with the above-mentioned group at all temperatures tested (3–10% and 2–19% to skin and intestinal mucus, respectively). In the case of skin mucus, three strains showed significant differences in adhesion depending on the incubation temperature, while two strains exhibited significant temperature-related differences in adhesion to intestinal mucus. The quartic polynomial fit was the best model to describe the changes in the adhesive ability related to the temperature changes. In conclusion, in order to optimize probiotic functionality in aquaculture, the dose may have to be adjusted with regard to the water temperature.