The impact of Spirulina, Chlorella and Phaeodactylum tricornutum (P. tricornutum) microalgal extracts obtained by pressurized liquid extraction (PLE) on antioxidant and anti-inflammatory activities, microbial growth and in vitro gut microbiota composition was evaluated. PLE, compared to conventional extraction, led to a significant (p < 0.05) increase in proteins, carbohydrates, polyphenols, and antioxidant capacities of the three microalgal extracts. Moreover, Spirulina and P. tricornutum extracts significantly (p < 0.05) reduced the in vitro activation of the inflammatory NF-κB pathway. The microalgal extracts had also an inhibitory effect on the pathogenic bacteria while potential beneficial Lactobacillus and Bifidobacterium strains increased growth. The effects of microalgal extracts on specific bacterial groups were analyzed by quantitative PCR technology, and bacterial gene copy numbers were affected by in vitro digestion process and colonic fermentation time. GC-MS results showed that microalgal biomolecules' digestion promoted the release of short-chain fatty acids (SCFAs) during in vitro colonic microbiota fermentation, particularly acetic, butanoic and propanoic, indicating that the biomolecules in microalgae extracts have potential health benefits for human gut.