A nutritional study was conducted to evaluate the inclusion of the green microalga Scenedesmus sp. at 5% (SCE-5) as an alternative fishmeal ingredient. This microalga was tested with four replicates during 45 days using isolipidic (18%), isoproteic (48%), and isoenergetic (1.9 MJ kg−1) diets. Fish fed Scenedesmus sp. showed similar growth and feed efficiency parameters as the control group. Regarding the digestive function, the SCE-5 diet enhanced the activity of alkaline pancreatic proteases, whereas it did not affect that of intestinal enzymes involved in nutrient absorption. No histological alterations were found in fish fed the SCE-5 diet, although a higher density of goblet cells in the anterior intestine and changes in gut microbiome diversity were found in this group, which collectively suggests positive effects of this green microalga on the intestine. Dietary Scenedesmus sp. improved the fillet’s nutritional quality in terms of n-3 polyunsaturated fatty acid (PUFA) levels, although it also increased its yellowish color. The overall results of this study showed that Scenedesmus sp. is a safe ingredient for compound feeds in rainbow trout when considering fish growth performance, animal condition, and health parameters, although it substantially affected the color of the fillet that may potentially affect consumers’ preferences.