This paper describes the evaluation of poloxamer-hydrogel biofilm-constructs for the routine efficacy testing of disinfectants at normal use strength. Aqueous solutions of poloxamer Pluronic F127 show thermo-reversible gelation, being liquid at temperatures <15 °C but firm gels at temperatures >15 °C. Chilled poloxamer solutions (30% w/v) were made up in a tryptone soy broth and inoculated with stationary-phase cultures of 14 foodborne spoilage microbes, including Pseudomonas, Bacillus, Staphylococcus, Micrococcus, enterobacteria and a yeast, as well as pathogen test-strains, including Listeria and Salmonella. Drops (either 200 µl or 100 µl) were placed onto pre-warmed, sterile, stainless steel discs held in sealed Petri dishes. The constructs were incubated for 5 h at 30 °C and all strains grew well in the poloxamer hydrogel. Incubated poloxamer gels and their discs were transferred to solutions of commercial disinfectant formulations containing either amphoteric surfactants, hydrogen peroxide with peracetic acid or silver ions, sodium hypochlorite, or alcohols with and without additives. After 5 min at 25 °C the test pieces were removed from the disinfectant solution and transferred to a neutraliser at 10–15 °C. These tests were carried out in triplicate. The gels dispersed rapidly, releasing the cells and enabling a count of the viable cells. All formulations effected a >5-log kill of planktonic challenges within 5 min. An effective killing of microbial cells within the biofilm-constructs was shown when the reduction was at least 0.3 log units. The results were highly reproducible, with patterns of susceptibility varying as a function of the organism, disinfectant type, and concentration. The experiments support the view that poloxamer hydrogels can be used for testing the disinfectant efficacy of various formulations against contaminants isolated from food and drink processes.