The efficacy of whole water column and subsurface applications of the biopesticide Zequanox®, a commercially prepared spray-dried powder formulation of Pseudomonas fluorescens (strain CL145A), were evaluated for controlling zebra mussels (Dreissena polymorpha) within 27-m2 enclosures in Lake Minnetonka (Deephaven, Minnesota). Five treatments consisting of (1) two whole water column Zequanox applications, (2) two subsurface Zequanox applications, and (3) an untreated control were completed on each of three independent treatment days during September 2014. The two types of samplers used in the study were (1) type 1 samplers, which were custom built multi-plate samplers (wood, perforated aluminum, and tile substrates) that were placed into Robinson’s Bay in June of 2013 to allow for natural colonization by zebra mussels, and (2) type 2 samplers, which consisted of zebra mussels adhering to perforated aluminum trays that were placed into mesh containment bags. One day prior to treatment, three individual samplers of each type were distributed to test enclosures and exposed to a randomly assigned treatment. Sampling to determine the zebra mussel biomass adhering to type 1 samplers and the survival assessments for zebra mussels contained in type 2 samplers were completed ~40 days after exposure. The zebra mussel biomass adhering to type 1 samplers and the survival of zebra mussels contained in type 2 samplers were significantly less in groups treated with the highest Zequanox concentrations and in groups that received whole water column applications than comparable groups treated with lower Zequanox concentrations and subsurface applications. However, standardization of biomass and survival results to the amount of Zequanox applied showed that the lower concentrations and subsurface applications were more cost efficient, with respect to product used, at reducing zebra mussel biomass and for inducing zebra mussel mortality. Although the subsurface application methods and lower treatment concentrations were more cost efficient, biological significance and management goals should be evaluated prior to selecting the application method. Development and refinement of additional application techniques may improve the utility of the subsurface Zequanox applications.