Virus-induced risks to water quality persist, with the viability of viruses in complex aquatic environments impacted by various factors, including coexisting bacteria and exposure to light. This study investigated the disinfection efficacy of UV254 irradiation and free chlorine (FC) on Bacteriophage phiX174 in the presence of coexisting bacteria, specifically Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). To elucidate the underlying mechanisms, this study examined the effects of bacterial compounds (i.e., E. coli lipopolysaccharide (LPS) and B. subtilis peptidoglycan (PGN)) on viral inactivation, thermal stability, and environmental persistence. The research results demonstrated that bacterial presence and their associated compounds significantly impaired viral inactivation, with greater concentrations of coexisting bacteria and their compounds exacerbating this effect. Additionally, the photoreactivation of phiX174 was explored following UV254 irradiation, indicating that high-dose UV254, coupled with low nutrient availability and minimal host bacteria levels, effectively suppressed viral photoreactivation. This study is the first to investigate the multifactorial effects on viral photoreactivation. The results offer novel insights into the challenges associated with virus-related pressures in water treatment, with potential implications for optimizing disinfection processes.