Chronic infections are commonly associated with biofilms formed by bacteria such as Staphylococcus epidermidis. With the increase in antibiotic resistant bacteria, maggot debridement therapy has been reintroduced for the treatment of chronic wounds. Studies have shown that the excretion/ secretions (ES) of Lucilia sericata larvae (maggots) contain many bioactive compounds which may contribute to the efficacy of maggot therapy. The present study evaluates the effect of L. sericata ES on the formation and disruption of S. epidermidis 1457 and 5179-R1 biofilms. These strains employ either polysaccharide intercellular adhesin (PIA) or accumulation associated protein (Aap) for intercellular adhesion. A semiquantitative biofilm assay was used to measure the formation/disruption of S. epidermidis 1457 and 5179-R1 biofilms by ES. ES activity was characterized according to concentration, incubation time and temperature, thermal stability, and size. Immunofluorescence microscopy was used to ascertain the effect of ES on PIA and Aap. In the presence of ES, S. epidermidis 1457 and 5179-R1 nascent biofilm formation was inhibited, and pre-formed biofilms disrupted. ES activity was temperature and time dependent, inactivated by heat treatment, and disruption depended on the mechanism of intercellular adhesion. The molecule(s) responsible was >10 kDa in size and appeared to have protease or glucosaminidase activity. ES interferes with S. epidermidis biofilm formation, specifically degrading factors employed in biofilm accumulation, which would increase bacterial susceptibility to antibiotics and the host's immune system. In purified form, ES-factors may have general applicability for the treatment or prevention of chronic biofilm infections caused by staphylococci.