Abstract This work mainly aimed to examine biofilm formation and prevention on linear low-density polyethylene (LLDPE) thermoplastic. LLDPE sheets with various levels of surface roughness, in a range of 0.50–8.50 (±0.05) μm, were incorporated with either 2-hydroxypropyl-3-piperazinyl-quinoline carboxylic acid methacrylate (HPQM) or silver-substituted zeolite (Zeomic). The results suggested that the surface roughness of LLDPE and the addition of HPQM or Zeomic agents had no significant effect on the mechanical properties and hydrophilicity of the LLDPE. The mechanism of Escherichia coli biofilm formation on LLDPE sheets involved an attachment and colonization of E. coli cells on LLDPE surface in the first day of incubation before the E. coli biofilm was fully developed on the 6th day, and finally detached from the LLDPE surface at the 9th day of incubation. HPQM and Zeomic could be used as E. coli inhibitors, but HPQM was more effective against E. coli than Zeomic. The recommended loading of HPQM for complete growth inhibition of E. coli on the LLDPE surface was 1500–2000 ppm. LLDPE with low surface roughness was found to be preferable if HPQM treatment was used. Physical schematics for migrations of HPQM and Zeomic molecules to prevent the biofilm formation on LLDPE surfaces were also proposed and discussed. The results of biofilm thickness were not necessarily correlated with the number of adhered viable cells. The results from maximum intensity projection (MIP) images and three-dimensional (3D) confocal images indicated that cell density in the biofilm matrix on LLDPE doped with HPQM increased with increasing incubation time, while cell density on neat LLDPE and LLDPE doped with Zeomic decreased with time. The biovolume of bacterial cells in the biofilm matrix also showed a correlation with the qualitative analysis of cell density observed by MIP and 3D confocal images.