Peritoneal dissemination of cancer cells is characteristic of advanced stages of ovarian, breast and lung cancers, and is associated with poor patient survival. The presence of cancer cells in effusions complicates treatment protocols, while cell eradication is seriously limited. One of the novel options available is cancer gene therapy with recombinant adeno-associated viruses. This combination represents the most promising gene delivery vehicles to neoplasmatic cells within serosal cavities due to their unique properties that include the ability to infect proliferating cells of broad host range, as well as the potential of long-term expression. Recombinant infectious adeno-associated virus serotype 2 particles (rAAV2) were produced in a helper-free system using an AAV-293 packaging cell line, and quantitatively analyzed by real-time PCR. Balb/c mice intraperitoneally pre-injected with L1 cancer cells were treated with different doses of rAAV2. Subsequently, the mice were sacrificed and intraperitoneal effusions were analyzed for rAAV presence and rAAV/β-galactosidase (LacZ) vector efficiency in order to infect cancer cells within the peritoneal cavity. We reported an efficient infection of L1 cancer cells disseminated into the peritoneal cavity by rAAV2. The expression of reporter genes (GFP and LacZ) attributable to the rAAV cell uptake was closely dependent on an applied multiplicity of infection ratio (MOI). The highest infection efficiency was observed at a MOI of 50 and 200. Our study confirmed the ability of adeno-associated viruses to facilitate gene transferability to cancer cells disseminated in the serosal cavity, as well as the potential usefulness of these viruses as a new approach in cancer gene therapy.