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Multidrug resistance (MDR) is a major global health challenge, necessitating alternative or adjuvant strategies such as lactoferrin (Lf) and probiotics. Opportunistic bacteria are key drivers of healthcare-associated infections (HAIs) due to their ability to form biofilms and cause persistent infections. Lf, a glycoprotein present in mammalian milk and secretions, exhibits antimicrobial properties as part of innate immunity. Similarly, probiotics—primarily lactic acid bacteria (LAB)—influence pathogen growth and adhesion. This study evaluated the antimicrobial and anti-biofilm effects of purified bovine Lf (bLf) versus commercial Lf, alone and combined with LAB-derived supernatants, against several opportunistic strains. Using qualitative and quantitative methods, we assessed the antimicrobial, anti-adherence, and immunomodulatory activities of bLf (Sigma-Aldrich), commercial Lf (Jarrow Formulas), and LAB supernatants. High-purity bLf demonstrated superior antimicrobial and antibiofilm effects compared to supplement-derived Lf. Furthermore, commercial Lf, combined with LAB metabolites, effectively inhibited biofilm formation, reduced inflammation, and prevented bacterial adhesion to eukaryotic cells. These findings support the potential use of lactoferrin, alone or in synergy with probiotics, as adjuvants or prophylactic agents, particularly for immunocompromised patients, to limit bacterial adherence and prevent chronic infections.