The efficacy and subsurface application of 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) custom built multi-plate samplers (type 1 samplers), with 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) samplers that were designed to contain zebra mussels (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 living 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 living 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 compared to groups treated with lower Zequanox concentrations and subsurface applications. However, standardization biomass and survival results to the amount of Zequanox applied showed that the lower Zequanox concentrations and subsurface applications were more efficient at reducing zebra mussel biomass and inducing zebra mussel mortality. Although more efficient, biological significance and management goals should be evaluated prior to selecting subsurface application methods and lower treatment concentrations for Zequanox applications. Development and refinement of additional application techniques may improve the utility of the subsurface Zequanox applications.