This study investigated the impact of storage duration on olive mill wastewater (OMW) quality and biological properties. Among two various olive varieties, Abani exhibited the highest phenolic compound concentration after 12 months of storage. Liquid chromatography-mass spectrometry (LC-MS) analysis identified 28 OMW constituents, with kampherol as the major one, and rosmarinic acid detected for the first time. Results showed significant differences in antioxidant and anti-inflammatory activities among samples. The inhibition of protein denaturation (IPD) test yielded the best value after 1 month for Abani OMW, while membrane-stabilizing potential (MSP) test values were highest after 12 months for the same variety. Anticoagulant activity, measured by activating partial thromboplastin time (APTT) and prothrombin time (PT), significantly varied with storage time, with the most favorable values found in Zlitni OMW immediately after olive oil extraction. These findings illuminate OMW changes over time, bearing significance for OMW management and potential applications.