, , , and are species of interest for commercial purposes due to their valuable nutritional profile. The aim of this study was to investigate the iron content in these four microalgae, with emphasis on their iron bioaccessibility assessed using an in vitro digestion system to simulate the process which takes place in the stomach and small intestine of dogs, followed by iron quantification using atomic absorption spectrometry. Furthermore, the extraction of soluble proteins was carried out and size exclusion chromatography was applied to investigate iron speciation. Significant differences ( < 0.004) in iron content were found between , which had the highest (1347 ± 93 μg g), and , which had the lowest (216 ± 59 μg g) iron content. , , and . showed an iron bioaccessibility of 30, 31, and 30%, respectively, while showed the lowest bioaccessibility (11%). The four species analysed presented soluble iron mainly bound to proteins with high molecular mass ranging from >75 to 40 kDa. showed the highest iron content associated with good bioaccessibility; therefore, it could be considered to be an interesting natural source of organic iron in dog nutrition.