Quantitative PCR (qPCR) was used to investigate whether largemouth bass virus (LMBV) can exist within biofilms. Suspended LMBV was partitioned into either laboratory-grown Pseudomonas fluorescens biofilms or pond-grown, mixed-population biofilms. Biofilm-entrapped LMBV retained infectivity when tested on epithelioma papillosum cyprini tissue culture cells. The LMBV associated with P. fluorescens biofilms were resistant to disinfection by sodium hypochlorite and an iodine-based compound (betadine) but were susceptible to ethanol. Largemouth bass virus was not detected in biofilms or water from ponds that had previously contained LMBV-positive fish, suggesting either that the viral concentrations were below the detection limit of qPCR or that the fish represented the main LMBV reservoir. This study illustrates the potential for LMBV to associate with bacterial biofilms and thereby gain protection from some chemical disinfectants.