Olive oil production yields a massive amount of byproduct, olive pomace (OP). Hexane-defatted Arbequina olive pomace from California, United States, was extracted with water and loaded to a preparative C18 chromatography. Phenolic desorption was applied by acidified methanolic-water gradients. Phenolic compound profiles and antioxidant/antimicrobial activities were determined. Results showed that the total phenolic contents of the fractions increased with the increase of the percentage of methanol in water gradients; however, the polar phenolic compound profiles generally decreased, while less-polar phenolic compound profiles increased. Oleuropein-aglycone-di-aldehyde (3,4-DHPEA-EDA) detected in water extract was not found in the acidified 35 mL/100 mL and acidified 70 mL/100 mL methanol fractions, but there was a new peak tentatively assigned as 3,4-DHPEA-EDA dimer. The in vitro antioxidant activities of water fractions were higher than that of higher methanolic fractions when they were compared at the same level of gallic acid equivalents; the same trend was observed for the antimicrobial activities evaluated using non-Shiga toxin-producing Escherichia coli O157:H7 and Listeria innocua. This study provides knowledge as data foundations for the practical valorization and industrial food applications of olive pomace extracts.