Discovering eco-friendly alternatives to synthetic chemicals has become an increasingly popular area of research. Natural products are now in the spotlight for their potential use as replacements for synthetic chemicals. To maximize the benefits of these natural products, it is important to use efficient extraction methods, especially from agroindustrial waste. Olive oil mill wastewater (OOMW) is a byproduct of the olive oil production process and is considered a pollutant; however, OOMW contains a wide range of phenolic compounds that have proven antimicrobial properties. This study investigates the extraction of these compounds from OOMW, with the aim of determining their potential antimicrobial activities against several bacterial strains and fungi, including Bacillus spizizenii, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella aerogenes, Streptococcus uberis, Enterococcus faecalis, and Candida albicans. The OOMW extracts (OEs) were prepared by using three different solvents: ethyl acetate, ethanol, and methanol. The highest total phenolic contents (4.03 g, GAE/L) and the strongest antibacterial activity were obtained with methanol extraction. All OEs showed no antifungal activity against C. albicans. OEs, particularly methanol extracts of OOMW, can be used as bioactive substances in various industries as nutraceuticals and food ingredients, respectively.